Process for inhibiting complement activation via the alternative pathway

ABSTRACT

A process of inhibiting activation of complement via the alternative pathway, including inhibiting the formation of complement activation products via the alternative pathway, is provided.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/918,349 filed Aug. 26, 1997, incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

[0002] The field of this invention is complement activation. Moreparticularly, the present invention pertains to a process for inhibitingcomplement activation via the alternative pathway, including forinhibiting the formation (i.e., generation or production) of complementactivation products via the alternative pathway.

BACKGROUND OF THE INVENTION

[0003] The complement system provides an early acting mechanism toinitiate and amplify the inflammatory response to microbial infectionand other acute insults. (Liszewski, M. K. and J. P. Atkinson, 1993, InFundamental Immunology, Third Edition. Edited by W. E. Paul. RavenPress, Ltd. New York). While complement activation provides a valuablefirst-line defense against potential pathogens, the activities ofcomplement that promote a protective inflammatory response can alsorepresent a potential threat to the host. (Kalli, K. R., P. Hsu, and D.T. Fearon, 1994, Springer Semin Immunopathol. 15:417-431; Morgan, B. P.,Eur. J. Clinical Investig. 24:219-228). For example, C3 and C5proteolytic products recruit and activate neutrophils. These activatedcells are indiscriminate in their release of destructive enzymes and maycause organ damage. In addition, complement activation may cause thedeposition of lytic complement components on nearby host cells as wellas on microbial targets, resulting in host cell lysis. The growingrecognition of the importance of complement-mediated tissue injury in avariety of disease states underscores the need for effective complementinhibitory drugs. No approved drugs that inhibit complement damagecurrently exist.

[0004] Complement can be activated through either of two distinctenzymatic cascades, referred to as the classical and alternativepathways. (Liszewski, M. K. and J. P. Atkinson, 1993, In FundamentalImmunology, Third Edition. Edited by W. E. Paul. Raven Press, Ltd. NewYork). The classical pathway is usually triggered by antibody bound to aforeign particle and thus requires prior exposure to that particle forthe generation of specific antibody. There are four plasma proteinsspecifically involved in the classical pathway: C1, C2, C4 and C3. Theinteraction of C1 with the Fc regions of IgG or IgM in immune complexesactivates a C1 protease that can cleave plasma protein C4, resulting inthe C4a and C4b fragments. C4b can bind another plasma protein, C2. Theresulting species, C4b2, is cleaved by the C1 protease to form theclassical pathway C3 convertase, C4b2a. Addition of the C3 cleavageproduct, C3b, to C3 convertase leads to the formation of the classicalpathway C5 convertase, C4b2a3b.

[0005] In contrast to the classical pathway, the alternative pathway isspontaneously triggered by foreign or other abnormal surfaces (bacteria,yeast, virally infected cells, or damaged tissue) and is thereforecapable of an immediate response to an invading organism (Liszewski, M.K. and J. P. Atkinson, 1993, In Fundamental Immunology, Third Edition.Edited by W. E. Paul. Raven Press, Ltd. New. York). There are fourplasma proteins directly involved in the alternative pathway: C3,factors B and D, and properdin (also called factor P). The initialinteraction that triggers the alternative pathway is not completelyunderstood. However, it is thought that spontaneously activated C3[sometimes called C3(H₂O)] binds factor B, which is then cleaved byfactor D to form a complex [C3(H₂O)Bb] with C3 convertase activity. Theresulting convertase proteolytically modifies C3, producing the C3bfragment, which can covalently attach to the target and then interactwith factors B and D and form the alternative pathway C3 convertase,C3bBb. The alternative pathway C3 convertase is stabilized by thebinding of properdin. Properdin extends its half-life six-to ten-fold(Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental Immunology,Third Edition. Edited by W. E. Paul. Raven Press, Ltd. New York).However, properdin binding is not required to form a functioningalternative pathway C3 convertase (Schreiber, R. D., M. K. Pangburn, P.H. Lesavre and H. J. Muller-Eberhard, 1978, Proc. Natl. Acad. Sci. USA75:3948-3952; Sissons, J. G., M. B. Oldstone and R. D. Schreiber, 1980,Proc. Natl. Acad. Sci. USA 77:559-562). Since the substrate for thealternative pathway C3 convertase is C3, C3 is therefore both acomponent and a product of the reaction. As the C3 convertase generatesincreasing amounts of C3b, an amplification loop is established(Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental Immunology,Third Edition. Edited by W. E. Paul. Raven Press, Ltd. New York). In asmuch as the classical pathway also may generate C3b, that C3b can bindfactor B and thereby engage the alternative pathway. This allows moreC3b to deposit on a target. For example, as described above, the bindingof antibody to antigen initiates the classical pathway. If antibodieslatch on to bacteria, the classical pathway generates C3b, which couplesto target pathogens. However, it has been suggested that from 10% to 90%of the subsequent C3b deposited may come from the alternative pathway(Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental Immunology,Third Edition. Edited by W. E. Paul. Raven Press, Ltd. New York). Theactual contribution of the alternative pathway to the formation ofadditional C3b subsequent to classical pathway initiation has not beenclearly quantified and thus remains unknown. Addition of C3b to the C3convertase leads to the formation of the alternative pathway C5convertase, C3bBbC3b.

[0006] Both the classical and alternative pathways converge at C5, whichis cleaved to form products with multiple proinflammatory effects. Theconverged pathway has been referred to as the terminal complementpathway. C5a is the most potent anaphylatoxin, inducing alterations insmooth muscle and vascular tone, as well as vascular permeability. It isalso a powerful chemotaxin and activator of both neutrophils andmonocytes. C5a-mediated cellular activation can significantly amplifyinflammatory responses by inducing the release of multiple additionalinflammatory mediators, including cytokines, hydrolytic enzymes,arachidonic acid metabolites and reactive oxygen species. C5 cleavageleads to the formation of C5b-9, also known as the membrane attackcomplex (MAC). There is now strong evidence that MAC may play animportant role in inflammation in addition to its role as a lyticpore-forming complex (Liszewski, M. K. and J. P. Atkinson, 1993, InFundamental Immunology, Third Edition. Edited by W. E. Paul. RavenPress, Ltd. New York).

[0007] Complement activation has been implicated as contributing to avariety of disease states and conditions, as well as complications froma variety of medical procedures (see references cited infra) such as:myocardial infarction; ischemia/reperfusion injury; stroke; acuterespiratory distress syndrome (ARDS); sepsis; burn injury; complicationsresulting from extracorporeal circulation (ECC) including most commonlyfrom cardiopulmonary bypass (CPB) but also from hemodialysis orplasmapheresis or plateletpheresis or leukophereses or extracorporealmembrane oxygenation (ECMO) or heparin-induced extracorporeal LDLprecipitation (HELP); use of radiographic contrast media; transplantrejection; rheumatoid arthritis; multiple sclerosis; myasthenia gravis;pancreatitis; and Alzheimer's disease. There is still no effectivecomplement inhibitory drug available for routine clinical use despitethe significant medical need for such agents.

[0008] The ability to specifically inhibit only the pathway causing aparticular pathology without completely shutting down the immune defensecapabilities of complement would be highly desirable. Based upon theavailable clinical data, it appears that in most acute injury settings,complement activation is mediated predominantly by the alternativepathway (Moore, F. D. 1994, Advan. Immunol. 56:267-299; Bjornson, A. B.,S. Bjornson and W. A. Altemeier, 1981 Ann. Surg. 194:224-231: Gelfand,J. A., M. Donelan, and J. F. Burke, 1983, Ann. Surg. 198:58-62). Thesefindings suggest that it would be advantageous to specifically inhibitalternative pathway-mediated tissue damage in a variety of acute injurysettings, for example, in myocardial infarction, ARDS, reperfusioninjury, stroke, thermal burns, and post-cardiopulmonary bypassinflammation. This would leave the classical pathway intact to handleimmune complex processing and to aid in host defense against infection.As essential components of the alternative pathway, factors B and D areattractive targets for specific inhibition of the alternative pathway.Because of its non-essential role, properdin, however, would not beexpected to be a suitable target for such intervention.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides a process of inhibitingalternative pathway complement activation. The process includes the stepof inhibiting properdin-induced stabilization of C3 convertase.Properdin-induced stabilization of C3 convertase is inhibited byinhibiting the binding of properdin to C3b or C3bBb. The binding ofproperdin to C3b is inhibited by exposing properdin to an effectiveamount of an antibody against properdin.

[0010] A process of the present invention is particularly useful ininhibiting complement activation via the alternative pathway in vivo insubjects, including humans, suffering from an acute or chronicpathological injury such as, but not limited to, myocardial infarction,acute respiratory distress syndrome, burn injury, stroke, multiplesclerosis, rheumatoid arthritis, Alzheimer's disease orischemia/reperfusion injury. In vivo inhibition of complement activationis accomplished by administering the anti-properdin antibody to thesubject. Pharmaceutical compositions containing anti-properdinantibodies are also provided.

[0011] The present invention provides, in one aspect, a process ofinhibiting the adverse effects of alternative complement pathwayactivation in a subject by administering to the subject an amount of ananti-properdin agent effective to selectively inhibit formation (i.e.,generation or production) of a complement activation product via thealternative complement pathway. Formation of such alternativepathway-dependent activation products refers to the generation orproduction of such products by complement activation, which productswhen generated or produced can be detected and include alternativepathway-dependent C3a, C5a, and/or C5b-9 (MAC) products. Ananti-properdin agent according to the invention blocks properdin asdescribed herein and selectively inhibits the formation of alternativecomplement pathway activation products. Such agents include ananti-properdin antibody, an antigen-binding fragment of ananti-properdin antibody, and a properdin-derived peptide. Preferably,the anti-properdin agent does not substantially activate Fcγ receptorsand/or the classical complement pathway.

[0012] The present invention provides, in another aspect, a process forinhibiting the adverse effects of classical complement pathwayactivation in a subject in which the classical complement pathway isinitiated by administering to the subject an amount of an anti-properdinagent effective to selectively inhibit formation of an alternativecomplement pathway activation product (e.g., alternativepathway-dependent C3a, C5a, MAC).

[0013] The present invention provides, in another aspect, a process forinhibiting the adverse effects of classical complement pathwayactivation in a subject in which the classical complement pathway isinitiated by administering to the subject an amount of an agent thatinhibits the alternative pathway C3 convertase effective to selectivelyinhibit formation of a complement activation product via the alternativecomplement pathway (e.g. alternative pathway-dependent C3a, C5a, MAC).

[0014] The present invention, in another aspect, provides a process forperforming a medical procedure on a subject comprising: (a) passingcirculating blood from a blood vessel of the subject, through a conduitand back to a blood vessel of the subject, the conduit having a luminalsurface comprising a material capable of causing at least one ofcomplement activation, platelet activation, leukocyte activation, orplatelet-leukocyte adhesion in the subject's blood; and (b) introducingan anti-properdin agent into the subject's bloodstream in an amounteffective to reduce at least one of complement activation, plateletactivation, leukocyte activation, or platelet-leukocyte adhesionresulting from passage of the circulating blood through the conduit,wherein step (a) occurs before and/or during and/or after step (b).Preferably, the anti-properdin agent reduces the alternativepathway-dependent conversion of complement component C3 into complementcomponents C3a and C3b, and/or the alternative pathway-dependentformation of C5b-C9, and/or the alternative pathway-dependent leukocyteactivation. Medical procedures including therapeutic proceduresaccording to the invention include extracorporeal circulationprocedures, including for example, cardiopulmonary bypass (CPB)procedures.

[0015] The present invention provides, in another aspect, an article ofmanufacture comprising packaging material and a pharmaceutical agent(i.e., pharmaceutical composition) contained within the packagingmaterial, wherein: (a) the pharmaceutical agent comprises ananti-properdin agent, the anti-properdin agent being effective forreducing at least one of complement activation, platelet activation,leukocyte activation, or platelet adhesion caused by passage ofcirculating blood from a blood vessel of a subject, through a conduit,and back to a blood vessel of the subject, the conduit having a luminalsurface comprising a material capable of causing at least one ofcomplement activation, platelet activation, leukocyte activation, orplatelet-leukocyte adhesion in the subject's blood; and (b) thepackaging material comprises a label which indicates that thepharmaceutical agent is for use in association with an extracorporealcirculation procedure. Articles of manufacture according to theinvention include labels that indicate that the anti-properdin agentsare for use in association with a cardiopulmonary bypass procedure.

[0016] The invention provides a use of an anti-properdin agent in thepreparation of a medicament for selectively inhibiting formation ofcomplement activation products via the alternative complement pathway ina subject in need thereof. Also provided is a use of an anti-properdinagent in the preparation of a medicament for selectively inhibitingformation of complement activation products via the alternativecomplement pathway in a subject in which the classical complementpathway is initiated. Additionally provided is a use of an alternativepathway C3 convertase-inhibiting agent in the preparation of amedicament for selectively inhibiting formation of complement activationproducts via the alternative complement pathway in a subject in whichthe classical complement pathway is initiated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In the drawings, which form a portion of the specification:

[0018]FIG. 1 shows binding of human properdin to C3b.

[0019]FIG. 2 shows the dose-dependent inhibition of properdin binding toC3b using an anti-properdin monoclonal antibody.

[0020]FIG. 3 shows the dose-dependent inhibition of properdin binding toC3bBb using an anti-properdin monoclonal antibody.

[0021]FIG. 4 shows the effects of human serum containing complementcomponents on the deposition of the membrane attack complex (MAC).

[0022]FIG. 5 shows the inhibition of membrane attack complex (MAC)deposition caused by an anti-properdin monoclonal antibody.

[0023]FIG. 6 shows the effects on an anti-properdin monoclonal antibodyon rabbit erythrocyte lysis.

[0024]FIG. 7 shows the inhibition of the formation of alternativecomplement pathway activation products, including C3a and MAC, using ananti-properdin monoclonal antibody in a tubing loop model ofcardiopulmonary bypass (CPB).

[0025]FIG. 8 shows the lack of Fcγ receptor activation as detected bylack of superoxide generation, using an F(ab)₂ fragment preparation ofan anti-properdin monoclonal antibody with properdin.

[0026]FIG. 9 shows the inhibition of the formation of alternativecomplement pathway activation products, including MAC, initiated byheparin-protamine complexes using an anti-properdin monoclonal antibody.

[0027]FIG. 10 shows the inhibition of the formation of alternativecomplement pathway activation products, including MAC, initiated byovalbumin/anti-ovalbumin immune complexes using an anti-properdinmonoclonal antibody.

[0028]FIG. 11 shows the inhibition of the formation of alternativepathway-dependent complement and leukocyte activation products,including inhibition of C3a, MAC or elastase-antitrypsin complex productformation, using an anti-properdin agent in an ex vivo model ofcardiopulmonary bypass (CPB).

DETAILED DESCRIPTION OF THE INVENTION

[0029] I. The Invention

[0030] Properdin is one of three unique plasma proteins that aredirectly involved in the alternative pathway. Together with factor D andfactor B, all three are potential targets for the development oftherapeutic agents to inhibit the alternative pathway. As set forthbelow, properdin is shown for the first time herein to be a suitabletarget for a process of inhibiting complement activation via thealternative pathway. Properdin is also shown for the first time hereinto be a suitable target even when the classical complement pathway hasbeen initiated.

[0031] Factor D is a serine proteinase with only a single known naturalsubstrate: factor B bound to C3b (Volanakis, J. E., S. R. Barnum, and J.M. Kilpatrick, 1993, Methods in Enzymol. 223:82-97). The serumconcentration of factor D, 2 μg/ml, is the lowest of any complementprotein (Liszewski, M. K. and J. P. Atkinson, 1993, In FundamentalImmunology, Third Edition. Edited by W. E. Paul. Raven Press, Ltd. NewYork). Factor D is known to be the rate limiting enzyme for thealternative pathway and therefore a suitable target for therapeuticmethods of inhibition complement activation via the alternative pathway.We believe, however, there are several reasons why inhibitors of factorD may not be ideal therapeutic agents for inhibiting complementactivation. Firstly, serine proteinases are also critically involved inthe coagulation and fibrinolytic systems, and it has proven difficult toidentify specific inhibitors of factor D (Kilpatrick, J. M., 1996, IBC'sSecond Annual Conference on Controlling the Complement System for NovelDrug Development, Conference Binder; Whitty, A., 1996, IBC's SecondAnnual Conference on Controlling the Complement System for Novel DrugDevelopment, Conference Binder). Secondly, factor D is a small protein(24.4 kDa) and is rapidly reabsorbed, and catabolized by the kidney witha fractional catabolic rate of 60% per hour. The steady state serumconcentration of factor D is maintained by a correspondingly high rateof synthesis. Therefore, it may be difficult to inhibit factor Dactivity in patient serum for prolonged periods without complicated drugdosing regimes. Thirdly, factor D is synthesized by adipocytes and thereis evidence from studies with genetically obese mice that factor D mayhave a regulatory role in fat metabolism (White, R. T., D. Damm, N.Hancock, B. S. Rosen, B. B. Lowell, P. Usher, J. S. Flier, and B. M.Speigelman., 1992, J. Biol. Chem. 267:9210-9213). Therefore, inhibitionof factor D may have detrimental secondary effects on patients that arenot directly related to complement inhibition.

[0032] Factor B plays a key role in the alternative pathway since itprovides the catalytic subunit, Bb, for the C3 convertase, C3bBb(Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental Immunology,Third Edition. Edited by W. E. Paul. Raven Press, Ltd. New York).Therefore, factor B also would appear to be another suitable target fortherapeutic methods of inhibiting complement activation via thealternative pathway. Alone, factor B is a zymogen with no knowncatalytic activity, but after binding C3b, the factor B serineproteinase can be activated by cleavage by factor D. Based on this, itshould be possible to develop specific inhibitors of factor Bb catalyticactivity as therapeutic agents to inhibit the alternative pathway.However, similar to the experience with factor D, it has provendifficult to identify inhibitors of factor Bb proteinase activity thatdo not also inhibit serine proteinases involved in blood coagulationhemostasis (Whitty, A., 1996, IBC's Second Annual Conference onControlling the Complement System for Novel Drug Development, ConferenceBinder). In any case, we believe factor B is probably not the besttarget for the development of therapeutic agents to inhibit thealternative pathway. Factor B is an abundant serum protein (−210 μg/ml)(Clardy, C. W., 1994, Infect. Immun. 62:4539-4555; Liszewski, M. K. andJ. P. Atkinson, 1993, In Fundamental Immunology, Third Edition. Editedby W. E. Paul. Raven Press, Ltd. New York) and it would probably requirea correspondingly high concentration of an inhibitor of factor B toeffectively block activation of the alternative pathway. Monoclonalantibodies to human factor B, however, have been prepared and tested fortheir in vitro ability to block alternative complement pathwayactivation by endotoxin (LPS) (Clardy, C. W. 1994, Infect Immun.62:4539-4555). One of the four monoclonal anti-factor B antibodiestested was able to effectively block alternative pathway activation. Theother three antibodies tested failed to block despite having affinitiesthat were similar to the blocking antibody. In three other studies ofanti-factor B monoclonal antibodies that were cited by Clardy, et al.,supra (1994), two monoclonals increased factor B activity by stabilizingthe alternative pathway convertase, one increased factor B activity byenhancing binding of B to C3b, three decreased factor B activity bydestabilizing the convertase and two decreased factor B activity byblocking binding of factor B to C3b.

[0033] Properdin plays a role in the regulation of the alternativepathway by virtue of its ability to bind and stabilize the inherentlylabile C3 and C5 convertase complexes (C3bBb and C3bBbC3b) (Nolan, K. F.and K. B. M. Reid, 1993, Methods Enzymol. 223:35-47), although the exactmechanism of C3 convertase stabilization is unknown (Daoudaki, M. E., J.D. Becherer and J. D. Lambris, 1988. J. Immunol. 140:1577-1580). Bindingof properdin to these complexes may result in a decreased rate ofdissociation of the Bb catalytic subunit and may also protect thecomplexes from degradation by the negative regulatory proteins, factorsI and H. However, properdin is not required for functional C3 convertaseactivity (Schreiber, R. D., M. K. Pangburn, P. H. Lesavre and H. J.Muller-Eberhard, 1978, Proc. Natl. Acad. Sci. USA 75:3948-3952; Sissons,J. G., M. B. Oldstone and R. D. Schreiber, 1980, Proc. Natl. Acad. Sci.USA 77:559-562; Pangburn, M. K. and H. J. Muller-Eberhard, 1984,Springer Semin. Immunopathol. 7:163-192). The concentration of properdinin normal human plasma was determined to be 4.3-5.7 μg/ml, making it oneof the least abundant complement proteins (Nolan, K. F. and K. B. M.Reid, 1993, Methods Enzymol. 223:35-47). In early studies, the plasmaconcentration of properdin was reported to be in the 20-25 mg/ml range;however, this estimate was based on an incorrect molar extinctioncoefficient for the protein. It is now known that the true plasmaconcentration of properdin is significantly lower (Nolan, K. F. and K.B. M. Reid, 1993, Methods Enzymol. 223:35-47). Human monocytes,neutrophils and T lymphocytes synthesize properdin (Schwaeble, W., W. G.Dippold, M. K. Schafer, H. Pohla, D. Jones, B. Luttig, E. Weihe, H. P.Huemer, M. P. Dierich, and K. B. M. Reid, 1993, J. Immunol.151:2521-2528; Schwaeble, W., U. Wirthmuller, B. Dewald, M. Thelen, M.K. Schafer, P. Eggelton, K. Whaley, and K. B. M. Reid, 1996, Molec.Immunol. 33(1):48; Schwaeble, W., H. P. Huemer, J. Most, M. P. Dierich,M. Strobel, C. Claus, K. B. M. Redi and H. W. Ziegler-Heitbrock, 1994,J. Eur. Biochem. 219:759-764). Unlike most other complement proteins,properdin does not appear to be synthesized by the liver. Properdin isstored in granules of human neutrophils and physiologically-relevantlevels of TNF, Il-8, fMLP and C5a induce its rapid secretion (Schwaeble,W., U. Wirthmuller, B. Dewald, M. Thelen, M. K. Schafer, P. Eggelton, K.Whaley, and K. B. M. Reid, 1996, Molec. Immunol. 33(1):48). In a humanmonocyte cell line, TNF and IL-1 enhanced both the abundance ofproperdin mRNA as well as secretion of the protein (Schwaeble, W., H. P.Huemer, J. Most, M. P. Dierich, M. Strobel, C. Claus, K. B. M. Redi andH. W. Ziegler-Heitbrock, 1994, J. Eur. Biochem. 219:759-764).

[0034] According to the present invention, of the three complementproteins that are uniquely involved in the alternative pathway,properdin is the most attractive target for development of apathway-specific complement inhibitor to treat acute inflammatorydisorders. As demonstrated hereinafter, an anti-properdin antibody thatprevents binding of properdin to C3 convertase totally inhibitsactivation of the alternative pathway. Therefore, as described for thefirst time herein, properdin is required for normal activation of thealternative pathway. Because properdin is demonstrated herein to play acentral role in complement activation, including in conditions involvinginitiation of the classical complement pathway, anti-properdin agentsmay be screened and selected that are unexpectedly effective inprocesses of selectively and potently inhibiting alternative complementpathway activation, including processes for inhibiting the formation ofcomplement activation products via the alternative pathway.

[0035] II. Process of Inhibiting Alternative Pathway Activation ofComplement

[0036] In one aspect, a process of the present invention provides forinhibition of complement activation via the alternative pathway,including for inhibiting the formation of complement activation productsvia the alternative pathway (e.g., MAC formation).

[0037] It is not well understood how properdin interacts with C3convertase, although the primary binding specificity of properdin hasbeen shown to be directed towards C3b, (Nolan, K. F. and K. B. M. Reid,1993, Methods Enzymol. 223:35-47). The properdin binding site on C3b hasbeen localized to residues 1402-1435 in the alpha chain of C3, as judgedby peptide inhibition studies (Daoudaki, M. E., J. D. Becherer and J. D.Lambris, 1988. J. Immunol. 140:1577-1580). The analysis of overlappingpeptides indicates that the site could be further refined to residues1424-1432 (Alzenz, J., J. D. Becherer, I. Esparza, M. E. Daoudaki, D.Avita, S. Oppermann, and J. D. Lambris, 1989, Complement Inflamm.6:307-314). There is also evidence that properdin binds to factor B andthis interaction appears to take place through sites on both the Ba andBb portions of the molecule. Although properdin binds cell-bound C3b,the binding is significantly enhanced with cell-bound C3bBb, suggestingthat binding sites of both C3b and Bb may contribute to the interactionof properdin with the convertase complex (Farries, T. C., P. J.Lachmann, and R. A. Harrison, 1988, Biochem. 252:47-54). Thus, properdinbinding to C3b can be inhibited whether the C3b is unconjugated orconjugated to factor B to form C3bBb (alternative pathway C3convertase). As shown in detail hereinafter in the Examples,anti-properdin antibodies can be screened and identified that blockproperdin binding to both C3b and C3bBb.

[0038] In accordance with a process of the present invention, properdinbinding to C3b is inhibited by exposing properdin, in the presence ofC3b, to an effective amount of an anti-properdin antibody, preferably ablocking antibody and most preferably a blocking antibody that lacks theability to active the Fcγ receptor upon binding to properdin, asdescribed herein. Means for determining an effective amount of anantibody are well known in the art. The anti-properdin antibody can be apolyclonal or monoclonal antibody. The use of monoclonal antibodies ispreferred. According to the invention, blocking antibodies againstproperdin have been identified and can be obtained from commercialsources (e.g., Quidel) or prepared using techniques well known in theart. Anti-properdin agents that are effective according to theinvention, are preferably antibodies that selectively block alternativepathway activation, including blocking formation of complementactivation products via the alternative pathway. However, in addition tosuch blocking antibodies, other blocking agents such as blockingpeptides that are demonstrated to substantially or totally inhibit thealternative pathway-dependent formation of C3a, C5a and/or MAC afterinitiation of the alternative pathway or classical pathway or both aresimilarly contemplated by the invention as described herein.

[0039] Polyclonal antibodies against properdin can be prepared byimmunizing an animal with properdin or an immunogenic portion thereof.Means for immunizing animals for the production of antibodies are wellknown in the art. By way of example, a mammal can be injected with aninoculum that includes properdin. Properdin can be included in aninoculum alone or conjugated to a carrier protein such as keyhole limpethemocyanin (KLH). Properdin can be suspended, as is well known in theart, in an adjuvant to enhance its immunogenicity. Sera containingimmunologically active antibodies are then produced from the blood ofsuch immunized animals using standard procedures well known in the art.

[0040] The identification of antibodies that immunoreact specificallywith properdin is made by exposing sera suspected of containing suchantibodies to properdin to form a conjugate between antibodies andproperdin. The existence of the conjugate is then determined usingstandard procedures well known in the art.

[0041] Properdin can also be used to prepare monoclonal antibodiesagainst properdin and used as a screening assay to identify suchmonoclonal antibodies. Monoclonal antibodies are produced fromhybridomas prepared in accordance with standard techniques such as thatdescribed by Kohler et al. (Nature, 256:495, 1975). Briefly, a suitablemammal (e.g., BALB/c mouse) is immunized by injection with properdin.After a predetermined period of time, splenocytes are removed from themouse and suspended in a cell culture medium. The splenocytes are thenfused with an immortal cell line to form a hybridoma. The formedhybridomas are grown in cell culture and screened for their ability toproduce a monoclonal antibody against properdin.

[0042] The inhibition of properdin binding to C3b is associated withinhibition of complement activation via the alternative pathway. Asshown in detail hereinafter in the Examples, anti-properdin agents,preferably anti-properdin antibodies not only blocked properdin bindingto C3b and C3bBb, but also blocked formation of products of thealternative pathway, including C5b-9, the Membrane-Attack Complex (MAC),which complex is the final end-product of complement activation. Stillfurther, the data in the Examples show that anti-properdin agents,preferably anti-properdin antibodies, also block alternativepathway-dependent erythrocyte lysis mediated by MAC. Still further, thedata in the Examples show that anti-properdin agents, preferablyanti-properdin antibodies and their antigen-binding fragments such asF(ab)₂, are effective in inhibiting alternative pathway complementactivation in models of cardiopulmonary bypass and in conditions ofclassical pathway complement activation.

[0043] The present findings are surprising and unexpected in view of theexisting literature. For example, Schreiber et al. demonstrated that thealternative pathway could be functionally assembled by mixing all of thealternative pathway proteins except properdin; it was concluded thatproperdin is not required for alternative pathway initiation andamplification. (Schreiber, R. D., M. K. Pangburn, P. H. Lesavre and H.J. Muller-Eberhard, 1978, Proc. Natl. Acad. Sci. USA 75:3948-3952).Moreover, alternative pathway activation initiated by measlesvirus-infected HeLa cells in the absence of IgG was the same in theabsence or presence of properdin. (Sissons, J. G., M. B. Oldstone and R.D. Schreiber, 1980, Proc. Natl. Acad. Sci. USA 77:559-562). These priorfindings have led to the generally accepted hypothesis that “properdinis not an essential component for the activation of the pathway, but itspresence does result in more rapid amplification of bound C3b” [emphasisadded] (Pangburn, M. K. and H. J. Muller-Eberhard, 1984, Springer Semin.Immunopathol. 7:163-192). These references effectively teach away fromthe identification of properdin as a target for alternative pathwayintervention. In contrast, according to the present invention, properdinis now identified as an essential component of and required foralternative complement pathway activation. Thus, a process of thepresent invention relates to the selective inhibition of alternativepathway by an anti-properdin agent. Such an agent surprisingly andeffectively blocks the alternative complement cascade, including inconditions involving initiation of the classical complement pathway.

[0044] III. Process of Treating Pathological Injury

[0045] The ability to inhibit complement activation using a process ofthe present invention provides a therapeutic regimen for treatment ofpatients having clinical symptoms in which complement activation isdeleterious. The complement system has been implicated as contributingto the pathogenesis of numerous acute and chronic disease states andconditions, as well as complications from a variety of medicalprocedures, including myocardial infarction (Moroko, P. R., C. B.Carpenter, M. Chiarello, M. C. Fishbein, P. Radva, J. D. Knostman, andS. L. Hale, 1978, Lab Invest. 48:43-47; Kilgore, K. S., G. S.Friedrichs, J. W. Homeister, and B. R. Lucchesi, 1994, Cardiovasc. Res.28:437-44; Weisman, H. F., T. Bartow, M. K. Leppo, H. C. Marsh, G. R.Carson, M. F. Concino, M. P. Boyle, K. H. Roux, M. L. Weisfeldt; and D.T. Fearon, 1990, Science 249:146-151; Schafer, P. J., D. Mathey, F.Hugo, and S. Bhaki, 1986, J. Immunol. 137:1945-1949), stroke(Kaczorowski, S. L., J. K. Schiding, C. A. Toth and P. M. Kochanek,1995, J. Cereb. Blood Flow Metab. 15:860-864; Morgan, B. P., P. Gasque,S. K. Singhrao, and S. J. Piddlesden, 1997, Exp. Clin. Immunogenet. 14:19-23; Vasthare, U. S., R. H. Rosenwasser, F. C. Barone, and R. F. Tuma,1993, FASEB J. 7:A424-429) ARDS (Mulligan, M. S., C. W. Smith, D. C.Anderson, R. F. Todd, M. Miyaska, T. Tamatani, T. B. Issekuts, and P. A.Ward, 1993, J. Immunol. 150:2401-2406; Solomkin, J. S., L. A. Cotta, P.S. Satoh, J. M. Hurst, and R. D. Nelson, 1985, Surgery 97:668-678;Mulligan, M. S., C. G. Yeh, A. R. Rudolph, and P. A. Ward, 1992, J.Immunol. 148:1479-1485; Anner, H., R. P. Kaufman, L Kobzik, C. R.Valeri, D. Shepro, and H. B. Hechtman, 1987, Ann. Surgery 206:642-648;Zilow, G., A. Sturm, U. Rother; and M. Kirschfink, 1990, Clin. Exp.Immunol. 79:151-157), reperfusion injury (Hsu, P., R. Simpson, T. F.Lindsay, T. Hebell, L. Kobzik, F. D. Moore, D. T. Fearon, and H. B.Hechtman, 1993, Clin. Res. 41:233A; Lindsay, T. F., J. Hill, F. Ortiz,A. Rudolph, C. R. Valeri, H. B. Hechtman, and F. D. Moore, 1992, Ann.Surg. 216: 677-683; Rubin, B. B., A. Smith, S. Liauw, D. Isenman, A. D.Romaschin and P. M. Walker, 1990, Am. J. Physiol. 259:H525-H531; Hill,J., T. F. Lindsay, F. Ortiz, C G Yeh, H. B. Hechtman, and F. D. Moore,1992, J. Immunol 149:1723-1729; Mulligan, M. S., E. Schmid, B.Beck-Schimmer, G. O. Till, H. P. Friedl, R. B. Brauer, T. E. Hugli, M.Miyasaka, R. L. Warner, K. J. Johnson, and P. A. Ward, 1996, J. Clin.Invest. 98:503-512; Pemberton, M., G. Anderson, V. Vetvicka, D. E.Justus, and G. D. Ross, 1993, J. Immunol. 150:5104-5113), sepsis/septicshock (Hack, C. E., J. H. Nuijens, R. J. F. Felt-Bersma, W. O.Schreuder, A. J. M. Eerenberg-Belmer, J. Paardekooper, W. Bronsveld, andL. G. Thijs, 1989, Am J Med 86:20-26; Bengston, A., and M. Heideman,1988, Arch. Surg. 23:645:649; Stevens, J. H., P. O'Hanley, J. M.Shapiro, F. G. Mihn, P. S. Satoh, J. A. Collins and T. A. Raffin, 1986,J. Clin. Invest. 77:1812-1816; Wakabayashi, G., J. A. Gelfand, W. K.Jung, R. J. Connolly, J. F. Burke, and C. A. Dinarello, 1991, J. Clin.Invest. 87:1925-1935), thermal burns (Solomkin, J. S., R. D. Nelson, D.E. Chenoweth, L. D. Solem and R. L. Simmons, 1984, Ann. Surg. 200,742-746; Bjornson, A. B., S. Bjornson and W. A. Altemeier, 1981, Ann.Surg. 194:224-231; Gelfand, J. A., M. Donelan, and J. F. Burke, 1983,Ann. Surg. 198:58-62; Gelfand, J. A., M. Donelan, and J. F. Burke, 1982,J. Clin. Invest. 70:1170-1176), post-cardiopulmonary bypass inflammation(Salama, A., F. Hugo, D. Heinrich, R. Hoge, R. Miller, V. Keifel, C.Muller-Eckhardt, and S. Bakdi, 1988, N. Engl. J. Med. 318:408-414;Chenoweth, D. E., S. W. Cooper, T. E. Hugli, R. W. Stewart, E. H.Blackstone and J. W. Kirklin, 1981, N. Engl. J. Med. 304:497-503; Moore,F. D., K. G. Warner, S. Assoussa, C. R. Valeri, and S. F. Khuri, 1987,Ann. Surg. 208:95-103; Rinder, C. S., H. M. Rinder, B. R. Smith, J. C.K. Fitch, M. J. Smith, J. B. Tracey, L. A. Matis, S. P. Squinto, and S.A. Rollins, 1995, J. Clin Invest. 96:1564-1572), hemodialysis (Hakim, R.M., J. Breillatt, J. M. Lazarus, and F. K. Port, 1984, New Eng. J. Med.311:878-882), use of radiographic contrast media (Arroyaue, C. M., andE. M. Tan, 1977, Clin. Exp. Immunol. 29:89-94), transplant rejection(Pruitt, S. K., W. M. Baldwin, H. C. Marsh, S. Lin, C. G. Yeh, and R. R.Bollinger, 1991, Transplantation 52:868-873; Leventhal, J. R., A. P.Dalmasso, J. W. Cromwell, J. L. Platt, C. J. Bolman, and A. J. Matas,1993, Transplantation 55:857-865; Dalmasso, A. P., G. M. Vercelloti, R.J. Fischel, R. J. Bolman, F. H. Bach and J. L. Platt, 1992, Am. J.Pathol. 140:1157-1166; Xia, W., D. T. Fearon, F. D. Moore, F. J. Schoen,F. Ortiz and R. L. Kirkman, 1992, Transplant Proc. 24:479-480),rheumatoid arthritis (Mollnes, T. E., T. Lea, O. J. Mellbye, J. Pahle,O. Grand, and M. Harboe, 1986, Arth. Rheum. 29:715-721; Morgan, B. P.,R. H. Daniels, and B. D. Williams, 1988, Clin. Exp. Immunol.73:473-478), multiple sclerosis (Linington, C., B. P. Morgan, N. J.Scolding, S. Piddlesden, and P. Wilkins, 1989, Brain. 112:895-911;Sanders, M. E., C. L. Koski, D. Robbins, M. L. Shin, M. M. Frank, and K.A. Joiner, 1986, J. Immunol. 135:4456), myasthenia gravis (Biesecker, G.and C. M. Gomez, 1989, J. Immunol. 142:2654-9; Nakano, S., and A. G.Engel. 1993 Neurology 43:1167-72), pancreatitis (Roxvall, L., A.Bengtson, and M. Heideman, 1989, J. Surg. Res. 47:138-143; Roxvall, L.,A. Bentston, and M. Heidman, 1990 Arch. Surg. 125:918-921) andAlzheimer's disease (Eikelenboom, P., C. E. Hack, J. M. Rozemuller, andF. C. Stam, 1989, Virchows Arch. (Cell pathol.) 56:259-62; Rogers, J. N.R. Cooper, and S. Webster, 1992, Proc. Natl. Acad. Sci. USA89:10016-20). While complement may not be the only cause of thepathogenesis in these conditions, it is nevertheless a majorpathological mechanism and represents an effective point for clinicalcontrol in many of these disease states.

[0046] Complement activation products have been detected in biologicalfluids or diseased tissues isolated from patients with many of theaforementioned conditions, and a correlation between the severity of theclinical indication with the abundance of complement activation productshas been demonstrated for some diseases (Zilow, G., A. Sturm, U. Rother,and M. Kirschfink, 1990, Clin. Exp. Immunol. 79:151-157; Hack, C. E., J.H. Nuijens, R. J. F. Felt-Bersma, W. O. Schreuder, A. J. M.Eerenberg-Belmer, J. Paardekooper, W. Bronsveld, and L. G. Thijs, 1989,Am. J. Med. 86:20-26; Gelfand, J. A., M. Donelan, and J. F. Burke, 1983,Ann. Surg. 198:58-62). The most compelling evidence directly implicatingcomplement in the pathogenesis of a diverse group of human diseasescomes from studies using art accepted animal models of such diseases. Insuch animal models, removal of classical pathway-activating antibodies(Fischel, R. J., R. M. Bolman, J. L. Platt, K. S. Naharian, F. H. Bach,and J. J. Matas, 1990, Trans. Proc. 22:1077-1083; Platt, J. L., R. J.Fischel, A. J. Matas, S. A. Reif, R. M. Bolman., and F. H. Bach, 1991,Transplantation 52:214-230), depletion of complement by cobra venomfactor (Moroko, P. R., C. B. Carpenter, M. Chiarello, M. C. Fishbein, P.Radva, J. D. Knostman, and S. L. Hale, 1978, Lab. Invest. 48:43-47;Mulligan, M. S., C. W. Smith, D. C. Anderson, R. F. Todd, M. Miyaska, T.Tamatani, T. B. Issekuts, and P. A. Ward, 1993, J. Immunol.150:2401-2406; Gelfand, J. A., M. Donelan, and J. F. Burke, 1983, Ann.Surg. 198:58-62; Leventahal, J. R., A. P. Dalmasso, J. W. Cromwell, J.L. Platt, C. J. Bolman, and A. J. Matas, 1993, Transplantation55:857-865), inhibition of complement activity (Weisman, H. F., T.Bartow, M. K. Leppo, H. C. Marsh, G. R. Carson, M. F. Concino, M. P.Boyle, K. H. Roux, M. L. Weisfeldt, and D. T. Fearon, 1990, Science249:146-151; Mulligan, M. S., C. G. Yeh, A. R. Rudolph, and P. A. Ward,1992, J. Immunol. 148:1479-1485; Pemberton, M., G. Anderson, V.Vetvicka, D. E. Justus, and G. D. Ross, 1993, J. Immunol.150:5104-5113), or testing in animals genetically deficient in specificcomplement components (Gelfand, J. A., M. Donelan, and J. F. Burke,1983, Ann. Surg. 198:58-62; Watson, W. C., and A. C. Townes, 1985, J.Exp. Med. 162:1878-1883), have all been shown to abrogate or delaypathogenesis.

[0047] Inherited deficiencies have been recognized in humans for nearlyevery complement component (Liszewski, M. K. and J. P. Atkinson, 1993,Fundamental Immunology, Third Edition. Edited by W. E. Paul. RavenPress, Ltd. New York). Deficiencies of components of the same pathwaycause similar clinical problems. Classical pathway componentdeficiencies (C1, C4, C2) commonly cause infections by a variety ofpyrogenic organisms and immune complex diseases, as does deficiency ofC3. Alternative pathway component deficiencies (P, D) often results inNeisserial infections. There is no evidence that properdin deficiencycauses increased susceptibility to immune complex disease or toinfections with organisms other than Neisseria. No homozygousdeficiencies in Factor B have been described (Morgan, B. P. and M. J.Walport, 1991, Immunology Today 12:301-306).

[0048] Inactivation of the alternative complement pathway isparticularly useful in subjects where activation is associated with thepathological effects of disease states. As set forth hereinbefore,complement activation is known to be associated with a large and diversegroup of disease states. Complement activation via the alternativepathway is particularly prominent in states of acute injury.

[0049] The complement-induced fulminant meningococcal septicemia inpatients with systemic meningococcal disease is likely causedpredominantly by activation of the alternative pathway (Brandtzaeg etal., Journal of Infectious Disease, 173:647-55, 1996). In patients withadult respiratory distress syndrome (ARDS), complement activationoccurred only via the alternative pathway for the first 48 hours (Zilowet al., J. Exp. Immunology, 79 151-57, 1990). An agent that suppressescomplement activation via both pathways has been used to treatpost-ischemic myocardial inflammation and necrosis in animal models ofcardiovascular disease (Weisman et al., Science, 249:146-71, 1990).According to Weisman et al., the acute tissue injury associated withnumerous autoimmune diseases is the result of complement activation.Complement induced tissue injury is found in immune complex-inducedvasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis,type II collagen-induced arthritis, and ischemia.

[0050] The role of the alternative complement pathway in inducingischemic cardiac damage during reperfusion has also been reported(Amsterdam et al., Amer. J. Physiol., 268: H448-H457, 1995). Mulligan etal. 1992, J. Immunol. 148:1479-1485 reported that complement activationplays a role in a variety of tissue injuries including glycogen-inducedperitonitis, lung and dermal injury after intra-alveolar or intra-dermaldeposition of IgG immune complexes, acute lung injury resulting fromintravascular activation of complement after injection of cobra venomfactor, and acute skin and lung injury after thermal trauma.

[0051] A variety of medical procedures utilize extracorporealcirculation (ECC), including hemodialysis, plasmapheresis,plateletpheresis, leukophereses, extracorporeal membrane oxygenation(ECMO), heparin-induced extracorporeal LDL precipitation (HELP) and mostcommonly cardiopulmonary bypass (CPB). These procedures expose blood orblood products to foreign surfaces that may alter normal cellularfunction and hemostasis. For example, it is well-established that CPBoften leads to complex inflammatory responses that result inpost-surgical complications, generally termed “post-perfusion syndrome”.Among these postoperative events are respiratory failure, bleedingdisorders, renal dysfunction and, in the most severe cases, multipleorgan failure (Wan, S., J-L. LeClerc, and J-L. Vincent, 1997, Chest112:676-692). The primary suspected cause of these CPB-related problemsis inappropriate activation of complement during the bypass procedure(Chenoweth, K., S. Cooper, T. Hugli, R. Stewart, E. Blackstone, and J.Kirklin, 1981, N. Engl. J. Med. 304:497-503; P. Haslam, P. Townsend, andM. Branthwaite, 1980, Anaesthesia 25:22-26; J. K. Kirklin, S. Westaby,E. Blackstone, J. W. Kirklin, K. Chenoweth, and A. Pacifico, 1983, J.Thorac. Cardiovasc. Surg. 86:845-857; Moore, F. D., K. G. Warner, B. A.Assousa, C. R. Valeri, and S. F. Khuri, 1988, Ann. Surg. 208:95-103; J.Steinberg, D. Kapelanski, J. Olson, and J. Weiler, 1993, J. Thorac.Cardiovasc. Surg. 106:1901-1918). While it appears that blood contactwith the tubing and oxygenator surfaces of the CPB circuit results inactivation of the alternative complement pathway (Chenoweth, K., S.Cooper, T, Hugli, R. Stewart, E. Blackstone, and J. Kirklin, 1981, N.Engl. J. Med. 304:497-503; Velthuis, H., P. G. M. Jansen, C. E. Hack, L.Eijsan, and C. R. H. Wildevuur, 1996, Ann. Thorac. Surg. 61:1153-1157),there is also evidence that the classical complement pathway isactivated during CPB (Wachtfogel, Y. T., P. C. Harpel, L. H. Edmunds,Jr. and R. W. Colman, 1989, Blood 73:468-471). Moreover, the classicalcomplement cascade is initiated after the termination of CPB due to theaddition of protamine to a patient's blood. Protamine is utilizedclinically to bind and remove the heparin that is added as ananti-coagulant during surgery. The heparin-protamine complexes causesignificant activation of the classical complement pathway (Steinberg,J., D. Kapelanski, J. Olson, and J. Weiler, 1993 J. Thorac. Cardiovasc.Surg. 106:1901-1918), further contributing to post-perfusion syndrome.

[0052] Activated complement species, particularly the anaphylotoxins C3aand C5a, are known to elicit a variety of inflammatory responses frommany cell types. For example, C5a can up-regulate cell adhesion moleculeexpression on neutrophils, and can also invoke lysosomal enzyme and freeradical release from both neutrophils and monocytes (Chenoweth, D. andT. Hugli, 1978, Proc. Natl. Acad. Sci. USA 75:3943-3947; Fletcher, M.P., G. Stakl, and J. Longhurst, 1993, Am. J. Physiol. 265:H1750-H1761).Likewise, C5a can activate platelets, rendering them incapable of normalclotting function (Foreman, K. E., A. A. Vaporciyan, B. K. Bonish, M. L.Jones, K. J. Johnson, M. M. Glovsky, S. M. Eddy, and P. A. Ward, 1994,J. Clin. Invest. 94:1147-1155). Finally, the terminal activatedcomplement product, C5b-9 (membrane-attack complex), can also affectplatelet and endothelial cell function (Foreman, K. E., A. A.Vaporciyan, B. K. Bonish, M. L. Jones, K. L. Johnson, M. M. Glovsky, S.M. Eddy, and P. A. Ward, 1994, J. Clin. Invest. 94:1147-1155; Hattori,R., K. K. Hamilton, R. D. Fugate, R. P. McEver, and P. J. Sims, 1989, J.Biol. Chem. 264:9053-9060). It is the actions of these complementspecies on neutrophils, platelets and other circulatory cells thatlikely lead to the various problems that arise after CPB.

[0053] Recently, there has been direct experimental evidence thatcomplement activation is, in fact, responsible for many of the changesinvolving dysfunction of the immune and hemostatic systems seen afterCPB. Using soluble complement receptor type 1 (sCR1), which preventsactivation of both the classical and alternative complement pathways,Gillinov, A. M., P. A. DeValeria, J. A. Winkelstein, I. Wilson, W. E.Curtis, D. Shaw, C. G. Yeh, A. R. Rudolph, W. A. Baumgartner, A.Herskowitz, and D. E. Cameron, (1993, Ann. Thorac. Surg. 55:619-624)demonstrated that inhibiting complement activation improved pulmonaryvascular resistance in pigs undergoing a CPB procedure. Utilizing an exvivo model of simulated CPB, Rinder et al. (1995, supra) showed thataddition of a monoclonal antibody to C5 significantly reduced theneutrophil and platelet activation that occurred during the bypassprocedure. The. C5 antibody blocks the cleavage of C5 in both theclassical and alternative complement pathways, and thus prevents theproduction of both the membrane-attack complex and the anaphylotoxin,C5a. The use of such an anti-C5 monoclonal antibody for reducingcomplement, platelet or leukocyte activation or platelet—leukocyteadhesion resulting from passage of a patient's blood via extracorporealcirculation is described in WO95/25540 [PCT/US95/03614].

[0054] IV. Pharmaceutical Compositions

[0055] A process of the present invention can thus be used to inhibitcomplement activation via the alternative pathway, including to inhibitthe formation of complement activation products via the alternativepathway, in patients by administering to a patient in need of complementinactivation an effective inhibiting amount of an anti-properdin agent,preferably an anti-properdin antibody or antigen-binding domain thereof.Preferably, the anti-properdin agent, most preferably an anti-properdinantibody or antigen-binding domain thereof, is administered in the formof a pharmaceutical composition.

[0056] Such a pharmaceutical composition comprises a therapeuticallyeffective amount of an anti-properdin agent, preferably ananti-properdin antibody formulated together with one or more non-toxicpharmaceutically acceptable carriers are disclosed. As used herein, theterm “pharmaceutically acceptable carrier” means a non-toxic, insertsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

[0057] The pharmaceutical compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, transdermally,topically (as by powders, ointments, or drops), bucally, or as an oralor nasal spray.

[0058] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils, (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral composition can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

[0059] Injectable preparations, for example, sterile injectable aqueousor oleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in preparation of injectables.

[0060] The injectables formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

[0061] In order to prolong the effect of a drug, it is often desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.Injection depot forms are made by forming micorencapsule matrices of thedrug, in biodegradable polymers such aspolylactide-polylactide-polyglycolide. Depending upon the ratio of drugto polymer and the nature of the particular polymer employed, the rateof drug release can be controlled. Examples of other biodegradablepolymers include poly (orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes of microemulsions which are compatible with body tissues.

[0062] Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carries such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

[0063] Solid compositions of a similar type may be employed as fillersin soft and hard-filled gelatin capsules using such excipients aslactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0064] The active compounds can also be in micro-encapsulated form withone or more excipients as noted above. The solid dosage forms oftablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings, release controllingcoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other inertdiluents, e.g., tableting lubricants and other tableting acids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

[0065] Dosage forms for topical or transdermal administration of acompound of this invention include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants or patches. The activecomponent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. Ophthalmic formulation, ear drops, eye ointments, powders andsolutions are also contemplated as being within the scope of thisinvention.

[0066] The ointments, pastes, creams and gels may contain, in additionto an active compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

[0067] Powders and sprays can contain, in addition to the compounds ofthis invention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicaters and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chloroflurohydrocarbons.

[0068] Transdermal patches have the added advantages of providingcontrolled delivery of a compound to the body. Such dosage forms can bemade by dissolving or dispensing the compound in the proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the compound in a polymermatrix or gel.

[0069] The Examples that follow illustrate preferred embodiments of thepresent invention and are not limiting of the specification and claimsin any way.

EXAMPLE 1

[0070] C3b-Properdin Binding

[0071] Polystyrene microtiter plates were coated with human C3b (0.5μg/50 μl per well) (Calbiochem, San Diego, Calif., Cat. No. 204860) inVeronal buffered saline (VBS): (5 mM diethyl barbiturate, 120 mM NaCl, 5mM MgCl₂, 5 mM EGTA) overnight at 4° C. After aspirating the C3bsolution, wells were blocked with VBS containing 0.5% human serumalbumin (HSA) (Sigma Chemical Company, St. Louis, Mo., Cat. No. A9511)for 2 hours at room temperature. Wells without C3b coating served asbackground controls. Aliquots of human properdin (or factor P) (AdvancedResearch Technology, San Diego, Calif., Cat. No. A139) at varyingconcentrations in blocking solution were added to the wells. Following a2 hour incubation at room temperature, the wells were extensively rinsedwith VBS.

[0072] C3b-bound properdin was detected by the addition of mousemonoclonal anti-human properdin antibody (detection antibody) (Quidel,San Diego, Calif., anti-human properdin monoclonal antibody P#2, Cat.No. A235) at 1:1000 dilution in blocking solution, which was allowed toincubate for 1 hour at room temperature. After washing the plates withVBS, a peroxidase-conjugated goat anti-mouse antibody (1:1000 dilutionin blocking solution) (Sigma Chemical Company) was added and allowed toincubate for 1 hour. The plate was again rinsed thoroughly with VBS, and100 μl of 3,3′,5,5′-tetramethyl benzidine (TMB) substrate (Kirkegaard &Perry Laboratories, Gaithersburg, Md., Cat. No. 50-65-00) was added.After incubation for 10 minutes at 25° C., the reaction of TMB wasquenched by the addition of 100 μl of phosphoric acid, and the plate wasread at 450 nm in a microplate reader (e.g., SPECTRA MAX 250, MolecularDevices, Sunnyvale, Calif.). The estimated K_(d) of properdin binding toC3b was based on the concentration of properdin at 50% maximal binding(Microcal Origin Program).

[0073] The ability of a murine anti-human properdin monoclonal antibody(blocking antibody) to inhibit C3b-P binding was evaluated by addingvarying concentrations of this antibody (blocking antibody, Quidel,anti-human properdin monoclonal antibody P#1, Cat. No. A233) to aconstant concentration of properdin (2 nM). The amount of properdinbound to C3b was detected with the antibody detection system describedabove.

[0074] As shown in FIG. 1, human properdin binds to C3b, which has beenimmobilized onto microtiter plate wells. The apparent binding constantfrom these data, defined as the concentration of properdin needed toreach half-maximal binding, is approximately 1 nM. When blockinganti-properdin monoclonal antibody is added along with properdin in thisassay, dose-dependent inhibition of properdin binding to C3b is observed(FIG. 2). The IC₅₀ value of 1 nM indicates that the antibody binds withhigh-affinity to properdin, thereby blocking its interaction with C3b.

EXAMPLE 2

[0075] C3b(Bb)-Properdin Binding

[0076] This assay was carried out as described in Example 1 above withsome changes. Briefly, microtiter wells were coated with C3b, washed,blocked and incubated with various concentrations of normal human serum(NHS) (Sigma Chemical Company, Cat. No. S1764) diluted in blockingsolution for 2 hours at room temperature. Under the conditions of thisbinding assay, factor B in serum will bind solid phase-bound C3b and,after cleavage by factor D in serum, generates C3bBb. Properdin is knownto bind C3bBb with high affinity (Farries, T. C., P. J. Lachmann, and R.A. Harrison, 1988, Biochem. 252:47-54). Uncoated wells served asbackground controls. After washing, bound-properdin was detected withthe anti-properdin antibody as described in Example 1 above.

[0077] To evaluate the effect of the blocking antibody to inhibitproperdin binding to C3b(Bb), various concentrations of the blockingantibody described in Example 1 above were added to a fixedconcentrations of serum (4% in blocking solution). The amount ofproperdin bound to C3b(Bb) was detected using the detection systemdescribed in Example 1 above.

[0078] In addition to purified properdin, properdin in serum can bind toimmobilized C3b. Since serum also contains factor B, which binds C3bwith a resulting conversion to Bb after cleavage by factor D, it islikely that serum-derived properdin is binding the C3bBb complex in thisassay format. When the blocking anti-properdin monoclonal antibody ofExample 1 is added with human serum to C3b-coated wells, there is adose-dependent inhibition of properdin binding to C3bBb (FIG. 3). Again,the IC₅₀ value in this assay is 1-2 nM, consistent with the resultsobtained in FIG. 2 described in Example 1 above.

EXAMPLE 3

[0079] Alternative Pathway-Dependent MAC Assay:

[0080] The binding data of Examples 1 and 2 above reveal that theproperdin monoclonal antibody prevents the binding of properdin to C3band the functional C3 convertase (C3bBb). Since the literature suggeststhat properdin stabilizes the C3 convertase, it was of interest to us todetermine whether the properdin antibody might appreciably affect theterminal aspects of the alternative complement cascade. The final endproduct of this pathway is the C5b-9 membrane-attack complex (MAC). Toanalyze the effects of the properdin antibody on MAC formation via thealternative pathways, an assay was utilized in which bacterial LPS wasused as a substrate to initiate the alternative complement pathwaycascade.

[0081] Previous studies have demonstrated that lipopolysaccharide (LPS)from Salmonella typhosa (S. Typhosa) (Sigma Chemical Company, Cat. No.6386) serves as a potent substrate for complement alternative pathwayactivation (Clardy, C. W., 1994, Infect. Immun. 62:4539-4555).Microtiter wells were coated with LPS (2 μg/50 μl per well) in VBSovernight at 4° C. Uncoated wells served as background controls. Afteraspirating the LPS solution, wells were treated with blocking solutionand incubated with various concentrations of normal human serum.Following a 3 hour incubation at 37° C., deposited MAC was detected withmouse anti-human soluble C5b-9 monoclonal antibody (Quidel, Cat. No.A239) using standard ELISA methodologies essentially as described in theExamples above. The effect of the blocking antibody on the MAC formationwas evaluated by adding various concentrations of blocking antibody to afixed concentration of serum (4% in blocking solution). The amount ofinhibition of soluble C5b-9 formation was determined using the antibodydetection system described in the Examples above.

[0082] As demonstrated in FIG. 4, addition of increasing amounts ofnormal human serum, which contains all of the complement components,resulted in increased MAC deposition on the LPS surface. The formationof MAC in this assay could be completely prevented by the addition ofthe properdin monoclonal antibody, as seen in FIG. 5. These dataindicate that properdin does not merely stabilize and alter the kineticsof the alternative pathway, as suggested in the literature, butdemonstrates for the first time that properdin is in fact necessary forprogression of the cascade.

EXAMPLE 4

[0083] Alternative Pathway—Dependent Hemolysis

[0084] To confirm and extend these results, the properdin antibody wasexamined in another assay of the alternative pathway. Rabbiterythrocytes initiate the alternative complement cascade, and theresulting formation of MAC causes lysis of these cells. If the properdinantibody is capable of complete inhibition of the alternative pathway,then addition of the reagent to rabbit erythrocytes bathed in humanserum should prevent cellular lysis. This can be assayed by examiningthe light scattering caused by intact red blood cells; lysed cells donot diffract light, and there is a consequent reduction in scatteredlight. It is well established that rabbit erythrocytes specificallyactivate the complement alternative pathway, with a resulting lysis ofthe cells by the C5b-9 complex (Nolan, K. F. and K. B. M. Reid, 1993,Properdin. Methods Enzymol. 223:35-47). Normal human serum, at variousconcentrations in Gelatin Veronal Buffer (GVB) (Advanced ResearchTechnology) with 5 mM MgCl₂ and 10 mM EGTA, was incubated with 37° C.with a fixed number of rabbit erythrocytes (Advanced ResearchTechnology). A progressive decrease in light scatter (due to lysis ofintact cells) was measured at 595 nm as a function of time in atemperature-controlled ELISA plate reader (Polhill et al., J. Immunol.121:383:370). To determine the ability of blocking antibody to inhibithemolysis of rabbit erythrocytes, various concentrations of the blockingantibody were added to a fixed concentration of normal human serum (8%)and the assay was performed as described above. The data were recordedand analyzed with a SpectraMax plate reader and software.

[0085] As shown in FIG. 6, addition of serum in the absence of properdinantibody resulted in lysis of the cells and a dramatic reduction inlight scattering. Addition of increasing concentrations of the antibodycaused a decrement in erythrocyte lysis, with 30 nM antibody completelyblocking MAC-mediated cellular destruction. These results confirm thatmonoclonal antibodies that bind and block properdin interaction with C3convertase are potent reagents that can completely abrogate the effectsof the alternative complement pathway.

[0086] Skilled artisans recognize and accept that in vitro studies ofcomplement are representative of and predictive of the in vivo state ofthe complement system. By way of example, the use of in vitro ELISA(enzyme-linked immunosorbent assay) procedures to detect properdinassociated with lipopolysaccharide (LPS) is a “simple, rapid andreliable method for the assessment of complement function particularlythe detection of complement deficiency states” (Fredrikson et al., J.Immunol. Meth., 166:263-70, 1993). The authors conclude that the invitro technique can be used in vivo with the same likelihood of successin detecting alternative complement pathway activation in diseasestates.

[0087] Similarly, the use of a 34-amino acid peptide to study properdinbinding with C3b using an in vitro hemolysis test was found to be anappropriate indication of both the role of properdin during infectionand the mechanism of C3 convertase stabilization (Daoudaki et al., J. ofImmun., 140:1577-80, 1988).

[0088] Still further, the standard rabbit erythrocyte hemolysis assay(described in detail herein in Example 4), which assay is used tomeasure alternative complement pathway activity, is accepted in the artas being the “most convenient assay for the activity of the humanalternative pathway” (Pangburn, Meth. In Enzymology, 162:639-53, 1988).

EXAMPLE 5

[0089] Cardiopulmonary Bypass: Tubing Loop Model

[0090] To test the effect of a blocking anti-properdin monoclonalantibody, as described in Examples 1-4 above, on inhibition ofcomplement activation in cardiopulmonary bypass (CPB), a tubing loopmodel of CPB as described by Gong, J., R. Larsson, K. N. Edkahl, T. E.Mollnes, U. Nilsson, and B. Nilsson, 1996, J. Clin. Immunol. 16:222-229was utilized. Whole blood from a healthy donor was collected into a 7-mlvacutainer tube (Becton Dickinson, San Jose, Calif.) containing 20 U ofheparin/ml of blood. Polyethylene tubing like that used during CPB (PE330; I.D., 2.92 mm; O.D., 3.73 mm; Clay Adams, N.J.) was filled with 0.5ml of the heparinized human blood and closed into a loop with a shortpiece of silicon tubing. Heparinized blood containing 20 mM EDTA (whichinactivates complement) served as a background control. Sample andcontrol tubing loops were rotated vertically in a water bath for 1 hourat 37° C. After incubation, blood samples were transferred into 1.7 mlsiliconized eppendorf tubes which contained 0.5 M EDTA to give a finalEDTA concentration of 20 mM. The samples were centrifuged (4000×g for 5minutes at 4° C.) and the plasma was collected. The plasma samples werediluted to 10% with sample diluent buffer and the amounts of C3a as wellas soluble MAC (sMAC) were determined using ELISA assay kits followingthe manufacturer's instructions (Quidel, Catalog Nos. A015 for C3a andA009 for C5b-9/MAC). For complement inhibition studies, variousconcentrations (100-600 nM) of the blocking anti-properdin monoclonalantibody described in Examples 1-4 were added to the heparinized bloodimmediately before circulation for 1 hour at 37° C. Aftercirculation/rotation in a 37° C. water bath for 1 hour, aliquots wereanalyzed for soluble MAC and C3a as described above using ELISA assaykits (Quidel).

[0091] Using this simplified CPB paradigm in which standard CPB tubingwas partially filled with fresh human blood, leaving an air-bloodinterface and where the tubing is joined end-to-end with a siliconsleeve to form a loop, such that this blood-filled loop is rotated in aheated water bath (37°) to simulate the movement of blood through abypass circuit, there is marked activation of complement during therotation of the blood in the tubing. Importantly, the blockinganti-human properdin antibody causes significant inhibition of thiscomplement activation. This can be seen in FIG. 7, where the formationof soluble membrane-attack complex (sMAC) in the loop model is nearlycompletely inhibited by 100 nM anti-properdin antibody. Likewise, thesame amount of antibody causes a significant reduction in C3a formation(FIG. 7).

[0092] This is the first demonstration of the effectiveness of an agentthat selectively inhibits alternative pathway activation in a model ofCPB. In contrast, all prior agents to date that have been usedexperimentally to inhibit complement activation in CPB protocols inhibitboth the classical and alternative complement pathways (e.g., Gillinov,A. M., P. A. DeValeria, J. A. Winkelstein, I. Wilson, W. E. Curtis, D.Shaw, C. G. Yeh, A. R. Rudolph, W. A. Baumgartner, A. Herskowitz, and D.E. Cameron, 1993, Ann. Thorac. Surg. 55:619-624; Rinder, C. S., H. M.Rinder, B. R. Smith, J. C. K. Fitch, M. J. Smith, J. B. Tracey, L. A.Matis, S. P. Squinto, and S. A. Rollins, 1995, J. Clin. Invest.96:1564-1572). Because it has been suggested that both the classical andalternative pathways are activated during CPB (Wachtfogel, Y. T., P. C.Harpel, L. H. Edmunds, Jr. and R. W. Colman, R. W., 1989, Blood73:468-471), these results with an alternative pathway targeting agentare particularly surprising.

EXAMPLE 6

[0093] Blocking Agents: Lack of Fcγ Receptor Activation

[0094] As described in Example 5 above, the blocking anti-properdinmonoclonal antibody potently inhibits soluble MAC and C3a generation ina tubing loop model of cardiopulmonary bypass. The proinflammatoryagents (C5a, C3a and sMAC) generated by complement activation are knownto activate leukocytes, platelets and endothelial cells. As a marker forneutrophil activation, serum levels of neutrophil elastase in wholeblood in the tubing loop model were also determined. As expected,elastase levels in blood samples incubated in the tubing loop wereincreased over levels in control samples. However, release of elastasewas not inhibited by the anti-properdin monoclonal antibody. Rather,there was an unexpected increase in serum elastase levels withincreasing antibody concentrations. If immune-complexes are generatedwhen the anti-properdin monoclonal antibody is added to blood containingproperdin, these immune-complexes could interact with Fcγ receptors onneutrophils resulting in cellular activation. Fcγ receptors and theiractivation have been reviewed by Ravetch, J. V. and J. P. Kinet, 1991,Ann Rev. Immunol. 9:457-492 and Hulett, M. D. and P. M. Hogarth, 1994,Adv. Immunol. 57:1-127.

[0095] Agents according to the present invention that selectivelyinhibit alternative complement pathway activation are preferably agentsthat do not activate Fcγ receptors, e.g., via immune complex formationwith their antigen. Such agents may be screened for their ability toactivate Fcγ receptors by a variety of assays known in the art. Sincesuperoxide generation is one of the classic responses of neutrophils andother phagocytes to activation via Fcγ receptors (RII), a specific assayfor superoxide release from cells in diluted whole blood was utilized toscreen and evaluate agents and the possible role of Fcγ receptors inneutrophil activation by such an agent. Monoclonal antibody agents aregenerally ineffective aggregating agents upon binding to their antigenand thus ineffective to activate Fcγ receptors via immune complexformation. However, Fcγ activation was detected for a blockinganti-properdin monoclonal antibody as follows.

[0096] A chemiluminescence assay for superoxide production in dilutedwhole blood was performed (Tose, M. F. and A. Itamedani, 1992, Am. J.Clin. Pathol. 97:566-573). Fresh blood was collected by a finger prickand rapidly diluted 350-fold into phenol red-free RPMI-1640 mediacontaining Pen-Strep which was buffered with 10 mM HEPES (pH 7.4) at 37°C. The media also contains 10 μM lucigenin (Sigma Chemical Co.), acompound which becomes chemiluminescent when reduced by superoxide.Potential stimulators of oxidative burst (e.g., PMA or C5a) or buffercontrols were added to duplicate 1 ml samples of the dilutedblood-lucigenin-RPMI media and incubated at 37° C. for 2-3 hours. Atregular intervals, chemiluminescence was monitored by transferring eachsample into a liquid scintillation counter (Packard Model 1900 TR)operated in a single photon mode to determine the “cpm”. Controlexperiments demonstrated that the specific “cpm” signal was completelyinhibited by addition of 100 μg/ml superoxide dismutase (Sigma ChemicalCo.) to the samples.

[0097] One important characteristic of the Fcγ receptor (RII) is that itis activated by binding of polymeric immune-complexes; monomeric IgG isineffective. Therefore, blood cells should have to be exposedsimultaneously to properdin and anti-properdin monoclonal antibody toallow immune-complex formation and consequent cellular activation viaFcγ receptors. Results from the chemiluminescence assay demonstratesthat superoxide levels in diluted blood samples are not significantlyelevated over control levels by addition of either 5 μg/ml properdinalone or 100 nM monoclonal antibody alone. However, simultaneousaddition of both 5 μg/ml properdin and 100 nM monoclonal antibody to thediluted blood samples results in a marked increase in superoxidegeneration over control levels, indicating that cell activation occursvia Fcγ receptors. C5a also activates the neutrophil oxidative burstresponse via C5a receptors, and samples containing 10 nM C5a werepositive controls in this assay. The anti-human properdin monoclonalantibody has a binding specificity for the human protein and does notbind rat properdin. Consistent with this specificity, incubation ofblood samples with 5 μg/ml rat properdin and 100 nM anti-humanmonoclonal antibody did not elicit an increase in superoxide generationover control levels. The results of this study indicate that neutrophilactivation is mediated via binding of properdin-monoclonal antibodyimmune-complexes to Fcγ receptors on cells, resulting in an increasedrelease of elastase upon addition of the monoclonal antibody to thetubing loop model.

[0098] There are several potential strategies that can be used in thedesign of agents according to the present invention that avoid Fcγreceptor interactions. For monoclonal antibody agents, one approach isto select the human γ4 IgG isotype during construction of a humanizedantibody. The γ4 IgG isotype does not bind Fcγ receptors. Alternatively,a monoclonal antibody agent can be genetically engineered that lacks theFc region, including for example, single chain antibodies andantigen-binding domains. Yet another approach is to chemically removethe Fc region of a monoclonal antibody using partial digestion byproteolytic enzymes, thereby generating, for example, antigen-bindingantibody fragments such as Fab or F(ab)₂ fragments. Such antigen-bindingantibody fragments and derivatives are similarly useful as potentinhibitors of alternative pathway complement activation.

[0099] In this study, proteolysis was utilized to remove the Fc regionof the blocking anti-properdin monoclonal antibody described in theExamples above, which is a murine IgG1. Specifically, a procedure forgenerating F(ab)₂ from murine IgG1 using ficin digestion (Mariani, M, M.Camagna, L. Tarditi and E. Seccamani, 1991, Mol. Immunol. 28:69-71) wasutilized. The progressive ficin-mediated cleavage of this IgG1 antibodyyielded a 116 kD species corresponding to F(ab)₂ and another species at32 kD corresponding to the cleaved Fc region. Ficin digestion conditionswere identified which resulted in the generation of F(ab)₂ at high yieldand the total absence of any detectable intact IgG band onCoomassie-stained SDS-PAGE gels.

[0100] The potency of this F(ab)₂ fragment as an inhibitor of complementactivation was compared to that of the intact anti-properdin monoclonalantibody, using the rabbit RBC hemolysis assay as described in Example4. The results showed that the ficin-digested monoclonal antibodypreparation containing the F(ab)₂ fragment has essentially the identicalinhibitory activity as the intact monoclonal antibody when both aretested at 3.3 nM (partial inhibition) or 6.7 nM (complete inhibition).In addition, these anti-properdin agents have essentially equivalentpotency as inhibitors of C3 and sMAC generation in the tubing loop modelof cardiopulmonary bypass described in Example 5.

[0101] As shown in FIG. 8, when the activity of F(ab)₂ and the intactantibody were compared in the superoxide generation assay using dilutedblood, addition of both intact monoclonal antibody (100 nM) andproperdin (5 μg/ml) to the diluted blood samples resulted in a markedincrease in superoxide generation over control levels. In comparison,superoxide generation in the diluted blood samples following addition ofboth F(ab)₂ (100 nM) and properdin (5 μg/ml) was substantially reducedand was similar to superoxide generation after addition of the F(ab)₂alone (FIG. 8). At later time points (>130 minutes), superoxidegeneration in both of the F(ab)₂ containing samples was slightly higherthan in the control samples (FIG. 8). However, since the F(ab)₂preparation was not purified to remove contaminating Fc or trace amountsof intact monoclonal antibody, small amounts of such contaminants couldgenerate a small residual response. Fc contaminants can be removed bystandard purification methods if desired. The results of this studydemonstrate that generation of an antigen-binding fragment such asF(ab)₂ can essentially eliminate activation of blood cells via bindingof Fcγ receptors.

[0102] It is of interest that this type of activation using ananti-properdin monoclonal antibody has not been noted with otheranti-complement monoclonal antibodies. For example, a monoclonalantibody to C5 has been shown to block the classical and alternativecomplement pathways where they converge at C5 (the terminal complementpathway) by blocking the cleavage of C5, thus preventing production ofMAC and C5a (WO95/25540 [PCT/US95/03614]; Rinder, et al., supra (1995)).

[0103] According to the present invention, preferred therapeutic agentsmay be screened and prepared that lack such Fcγ receptor activation andare particularly effective in processes for selective inhibition of theformation of alternative complement pathway activation products. Inaddition, agents according to the present invention are preferablyagents that are selective for their inhibition of formation ofalternative pathway activation products (i.e., do not inhibit classicalpathway components) and that do not activate the classical complementpathway. Immune complexes, in addition to activating cells via bindingto Fcγ receptors, can trigger the classical pathway of complementactivation by binding to complement component C1. To show that theclassical pathway of complement activation was not activated by theblocking anti-properdin monoclonal antibody described above, duplicatesamples of normal human serum (NHS) were incubated at 37° C. for 120minutes with or without 200 nM anti-properdin monoclonal antibody (30μg/ml). At 0, 30, 60, and 120 minutes, 50 μl aliquots were removed fromthe mixture and chelated by the addition of EDTA to a finalconcentration of 13 mM in order to stop all magnesium- andcalcium-dependent complement activation. The concentration of thecomplement activation product C3a was determined in all samples using anELISA kit following the manufacturer's instructions (Quidel, Catalog No.A015). None of the samples containing the anti-properdin monoclonalantibody had elevated C3a levels compared to corresponding controlsamples. These results demonstrate that blocking anti-properdinmonoclonal antibody does not trigger activation of the classicalpathway. Furthermore, these results suggest that the structuraldeterminants on immune complexes recognized by C1 are different thanthose recognized by Fcγ receptors. Preferred agents according to theinvention are therefore agents that do not substantially activate Fcγreceptors or the classical complement pathway as shown herein.

EXAMPLE 7

[0104] Classical Pathway Activation: Heparin—Protamine Complexes

[0105] Protamine addition to heparinized blood has been shown toactivate the classical complement pathway (Cavarocchi, N. C., H. V.Schaff, T. A. Orszulak, H. A. Homburger, W. A. Schnell, and J. R. Pluth,1985, Surgery 98:525-531. Thus, complement activation occurs not onlyduring blood circulation through tubing for a bypass circuit like thatutilized in Example 5 above, but also after the addition of protamine(to neutralize anticoagulant heparin) at the end of the CPB procedure.To evaluate the effect of protamine on generation of soluble MAC,heparinized blood was incubated in tubes at 37° C. for 60 minutes with100 μg/ml protamine either in the absence or presence of a blockinganti-properdin monoclonal antibody as described in Examples 1-5 above.Samples were processed and analyzed for sMAC generation using an ELISAkit following the manufacturer's instructions (Quidel, Catalog No.A009).

[0106] As shown in FIG. 9, the anti-properdin monoclonal antibodyinhibits complement activation initiated by heparin-protamine complexes,under conditions where fresh heparinized blood was incubated asdescribed above in tubes at 37° C. for 60 minutes with 100 μg/mlprotamine either in the absence of presence of 13, 66, 200 or 330 nManti-properdin monoclonal antibody and as detected by sMAC generation.

[0107] Because complement activation occurs not only during the movementof blood through a bypass circuit, but also after the addition ofprotamine upon completion of CPB, and because this latter activationinvolves the classical complement pathway (Cavarocchi, N. C., H. V.Schaff, T. A. Orszulak, H. A. Homburger, W. A. Schnell, and J. R. Pluth,1985, Surgery 98:525-531), it was unexpected that an alternativepathway-specific anti-properdin agent would attenuate or substantiallyinhibit the production of complement activation products triggered byheparin-protamine complexes as shown in FIG. 9.

[0108] Although it has been suggested that the alternative pathway mightcontribute somewhat to the classical pathway-initiated production ofterminal activation products because the alternative pathway C3convertase could, in theory, be assembled from C3b generated in theclassical cascade, there has been a general paucity of experimental dataaddressing this hypothesis. A relatively recent study has moredefinitively addressed the question of alternative pathway contributionto the classical pathway, particularly as it pertains to the role ofproperdin. Specifically, Fredrikson et al. (1993) examined the effect ofproperdin deficiency on the amount of MAC generated after activation ofthe classical pathway. Their results reveal that the absence ofproperdin in serum had no effect on classical pathway production of MAC.In contrast, depletion of classical pathway components (i.e., C1q, C2,C4) completely abolished MAC generation in their assay system. Theseresults indicate that inhibiting properdin action with an anti-properdinagent should have no effect on the production of activated complementspecies after initiation of the classical pathway by protamine-heparincomplexes. Further support for this interpretation is supplied by thework of Soderstrom, C., J. H. Braconier, D. Danielsson, and A. G.Sjoholm, 1987, J. Infect. Dis. 156:107-112, who showed that serumbactericidal reactions mediated via the classical complement pathwaywere not impaired in properdin-deficient serum. These resultsspecifically teach that inhibiting properdin action should have little,if any, effect on the production of complement activation proteins afterinitiation of the classical complement pathway. More generally, thesedata imply that the alternative pathway contributes negligibly to theclassical pathway-induced production of complement activation products.This more general interpretation is supported by the work of Clardy, C.W., 1994, Infect. Immun. 62:4549-4555, who reported that an antibody tothe alternative pathway-specific component, factor B, had no effect onclassical pathway complement activation.

[0109] In agreement with what is seen during clinical CPB and as shownabove, protamine addition to heparinized human blood causes significantcomplement activation, as measured by the production of sMAC (FIG. 9).Remarkably, addition of the anti-properdin antibody to the heparinizedblood prior to the addition of protamine results in nearly completeinhibition of sMAC formation (FIG. 9) and demonstrates that ananti-properdin agent is effective in reducing the post-perfusioncomplications associated with CPB since it is capable of inhibiting bothclassical and alternative complement pathways.

EXAMPLE 8

[0110] Classical Pathway Activation: Immune Complexes

[0111] The classical complement pathway is typically triggered by immunecomplexes, for example, an antibody bound to a foreign particle, andthus requires prior exposure to that particle for the generation ofspecific antibody. There are four plasma proteins involved in theinitial steps of the classical pathway: C1, C2, C4 and C3. Theinteraction of C1 with the Fc regions of IgG or IgM in immune complexesactivates a C1 protease that can cleave plasma protein C4, resulting inthe C4a and C4b fragments. C4b can bind another plasma protein, C2. Theresulting species, C4b2, is cleaved by the C1 protease to form theclassical pathway C3 convertase, C4b2a. Addition of the C3 cleavageproduct, C3b, to the C3 convertase leads to the formation of theclassical pathway C5 convertase, C4b 2 a3b. To evaluate the effect ofimmune complexes on the generation of C3a and soluble MAC, immunecomplexes were prepared by incubating rabbit anti-ovalbumin IgG (28 mg)(Biodesign, Kennebunk, Me.) and ovalbumin (0.67 mg) (Sigma ChemicalCompany) in 3 ml PBS for 3 day at 4° C. to allow maximum precipitation.Preliminary experiments demonstrated that this ratio of reagentscorresponds to the equivalence point for the antigen-antibody reaction.The precipitate was collected by centrifugation at 15,000 rpm for 5 minat 4° C. and washed 3 times by resuspension in 5 ml of PBS andrecentrifugation. The final precipitate was resuspended in PBS at 1mg/ml and frozen in aliquots at −70° C. SDS-PAGE analysis confirmed thatthe precipitate contains essentially only antibody and antigen.

[0112] To test the effect of an anti-properdin monoclonal antibody onconditions of complement activation where the classical pathway isinitiated by immune complexes, triplicate 50 μl samples containing 90%NHS were incubated at 37° C. in the presence of 10 μg/ml immune complex(IC) or PBS, and parallel triplicate samples (+/− IC) also contained 200nM anti-properdin monoclonal antibody during the 37° C. incubation.After two hour incubation at 37° C., 13 mM EDTA was added to all samplesto stop further complement activation and the samples were immediatelycooled to 5° C. The samples were stored at −70° C. prior to beingassayed for complement activation products (C3a and sC5b-9) using ELISAkits (Quidel, Catalog Nos. A015 and A009) following the manufacturer'sinstructions. The results of a sMAC assay are shown in FIG. 10.

[0113] Surprisingly, the addition of an anti-properdin monoclonalantibody to the serum prior to the addition of immune complexessubstantially inhibited (e.g., ≧50%) both C3a and sMAC formation(approximately 80% for sMAC as shown in FIG. 10). Similar to the resultsdescribed in Example 7 above with heparin-protamine complexes, it waslikewise unexpected that an alternative pathway-specific anti-properdinagent would attenuate or substantially inhibit the production ofactivated complement species after initiation of the classical pathwayby immune complexes as shown in FIG. 10.

EXAMPLE 9

[0114] Cardiopulmonary Bypass: Ex Vivo Extracorporeal Circulation

[0115] To further confirm the usefulness of anti-properdin agents,including anti-properdin antibody agents, in reducing complementactivation during CPB and other extracorporeal procedures, which involvepassing circulating blood from a blood vessel of a subject, through aconduit and back to a blood vessel of the subject, studies wereperformed in which freshly-collected human blood was passed through acircuit that is identical to that which is typically used duringsurgical procedures requiring pediatric extracorporeal circulation. AnF(ab)₂ anti-properdin agent prepared as described in Example 6 was usedin these studies.

[0116] Pediatric extracorporeal circuits were assembled using ahollow-fiber pediatric membrane oxygenator (Lilliput oxygenator),polyvinyl chloride tubing polycarbonate connectors, and a minimallyocclusive roller pump. Oxygenator and circuitry were primed with 400 mlof Plasmalyte. Blood (225 ml) was drawn from a healthy volunteer into atransfer pack containing 1000 U heparin which was then added to theextracorporeal circuit. For studies of complement inhibition duringextracorporeal circulation, a F(ab)₂ preparation of an anti-properdinmonoclonal antibody in PBS (−25 μg/ml of blood) was added to thetransfer pack immediately before addition of blood to the extracorporealcircuit. As blood was introduced to the reservoir via the prime port,225 ml of prime fluid was simultaneously withdrawn distal to theoxygenator outlet to yield a final circuit volume of 400 ml and a finalhematocrit of 20%. Blood was circulated with prime, and complete mixingwas accomplished within 2 minutes. A baseline sample was drawn anddesignated as time 0. The circuit was maintained at 37° C. for 30minutes, then cooled to 28° C. over five minutes and maintained at thattemperature for 60 minutes, after which it was rewarmed to 37° C. for anadditional 60 minutes. Blood samples were drawn at multiple times duringrecirculation. Serum samples were prepared by immediate centrifugation,and stored at −70° C. in aliquots until assayed for C3a or sMAC viaELISA kits following the manufacturer's instructions (Quidel: C3a kit,Catalog No. A015; sMAC kit, Catalog No. A009) or neutrophil elastaseusing an ELISA as described by Brower, M. S. and P. C. Harpel, 1983,Blood 61:842-849.

[0117] As shown in FIG. 11, an F(ab)₂ preparation of a blockinganti-properdin monoclonal antibody inhibits complement activation in anex vivo model of CPB where fresh human blood was pumped through apediatric bypass circuit either in the absence (closed circles) orpresence.(open circles) of an F(ab)₂ preparation of an anti-properdinmonoclonal antibody. As described above, blood samples were collected atvarious times during the bypass procedure and analyzed for sMAC, C3a orelastase-antitrypsin complexes.

[0118] For this study, the two bypass circuits that were utilized wereconnected to a common non-pulsatory pump such that blood flow wasidentical in the two systems. Whereas one circuit contained untreatedblood, the other contained blood to which the anti-properdin agent wasadded prior to the onset of circulation. In addition in this study, aF(ab)₂ preparation of the anti-properdin monoclonal antibody wasutilized to ensure that properdin-anti-properdin complexes did nottrigger cellular signal transduction events by binding to Fcγ receptors.This F(ab)₂ antibody fragment was prepared by proteolytic cleavage withficin as described in Example 6. Standard proteolytic methodologies aswell as standard recombinant methodologies may be used to prepareantibody-based proteins, including fragments, derivatives, single chainantibodies (SCA) and antigen-binding domains that lack the Fc portion ofthe immunoglobulin that allows binding to Fcγ receptors (Janeway, C. andP. Travers, Jr., 1994, Immunobiology: the Immune System in Health andDisease. pp 3:28-3:30. Garland Publishing, Inc., New York).Alternatively, the potential binding of therapeutic antibodies Fcγreceptors can be eliminated, if desirable or necessary, by utilizingantibodies of the γ4 sub-class of IgG, which do not interact with thesereceptors (Janeway, C. and P. Travers, Jr., supra (1994)).

[0119] As demonstrated in FIG. 11, the onset of blood flow in this exvivo model of CPB resulted in the rapid production of sMAC and C3a, withthe levels of these activated complement components increasing as afunction of bypass time. Likewise, blood levels of elastase-antitrypsincomplexes, a marker of neutrophil activation (Finn, A., S. Naik, N.Klein, R. J. Levinsky, S. Strobel, and M. Elliott, 1993, J. Thorac.Cardiovasc. Surg. 105:234-241), increased with circulation time in theCPB circuit (FIG. 9). It has been postulated that neutrophil activationduring CPB results from the binding of complement activation species tothese cells (Rinder, C. S., H. M. Rinder, B. R. Smith, J. C. K. Fitch,M. J. Smith, J. B. Tracey, L. A. Matis, S. P. Squinto, and S. A.Rollins, S. A., 1995, J. Clin. Invest. 96:1564-1572; Wan, S., J-L.LeClerc, and J-L. Vincent, 1997, Chest 112:676-692). The parallelcircuit containing blood treated with the F(ab)₂ anti-properdinmonoclonal antibody preparation showed essentially no complementactivation, as revealed by the virtual absence of sMAC and C3a at allbypass times. Importantly F(ab)₂ anti-properdin also caused a reductionin neutrophil activation, as demonstrated by the reduction in bloodelastase-antitrypsin complex levels (FIG. 11). These results confirm theresults obtained with the tubing loop model described in Example 5above. These results further demonstrate that an anti-properdin agentsthat lacks Fcγ receptor activation ability effectively reduces thecomplement activation and related cellular inflammatory events thatresult from extracorporeal circulation and subsequent protaminecomplexation of heparin.

EXAMPLE 10

[0120] Blocking Agents: Screening of Properdin-Derived Peptides

[0121] Several decapeptides of human properdin as described byFredrikson, et al., supra (1996) were prepared and tested for theirability to block the effects of alternative complement pathwayactivation as described for anti-properdin antibodies in the Examplesabove. Specifically, these properdin-derived peptides were assayed fortheir ability to inhibit MAC formation in an ELISA as described inExample 3. Peptide 1 consisting of amino acids 43-52 of properdin (1158M.W.), peptide 2 consisting of amino acids 48-57 of properdin (1320M.W.) and peptide 3 consisting of amino acids 73-82 of properdin (1309M.W.) each reduced MAC formation in this assay, with IC₅₀ values of 268μM, 335 μM and 242 μM, respectively. In contrast, peptide 4 consistingof amino acids 218-227 of properdin (1173 M.W.) did not similarlyinhibit MAC formation in this assay (IC₅₀>600 μM). When these fourpeptides were tested as described in Example 2 above, peptides 1, 2 and3, but not peptide 4, blocked C3bBb binding, with the IC₅₀ for the 3blocking peptides in the range of about 400-600 μM.

[0122] In a previous study by Fredrikson, et al., supra (1996) tocharacterize a dysfunctional properdin protein from a patient with TypeIII properdin deficiency, 87 overlapping decapeptides of humanproperdin, including the four peptides described above, weresynthesized. When these peptides assayed at concentrations of 0-200μg/ml for their ability to compete with the binding of properdin to C3bcoated plates, five peptides designated as 9, 10, 15, 44 (correspondingto peptides 1, 2, 3 and 4 herein) and 81 were determined by Fredrikson,et al., supra (1996) to compete with properdin for C3 binding. Thesepeptides were not tested in assays of complement activation, such as theMAC formation assay described above.

[0123] Since properdin has now been demonstrated according to thepresent invention to be a critical component for activation of thealternative pathway, anti-properdin agents, including properdin-derivedpeptides, may be screened, identified and selected for their ability toblock alternative pathway activation, as demonstrated herein, forexample, by blocking MAC formation. Since the screening assay showedthat inhibition of MAC formation was essentially complete at the higherpeptide concentrations, properdin was again demonstrated to be requiredfor activation of the alternative pathway. Anti-properdin agents,including properdin-derived peptides, may be identified according to thepresent invention as effective agents in a process for selectivelyinhibiting the generation (i.e., formation or production) of analternate complement pathway activation product in a subject in whicheither the alternative pathway or the classical pathway has beeninitiated, including in subjects with a variety of disease states andconditions, as well as complications from a variety of medicalprocedures, and including subjects with acute and/or chronicpathological injuries as described and referenced herein.

EXAMPLE 11

[0124] Blocking Agents: Screening and Identification

[0125] Agents, which selectively block the formation of complementactivation products via the alternative complement pathway, includingpreferred anti-human properdin antibodies, may be obtained and thenscreened, identified and selected as taught herein, for their ability tosubstantially or completely block the formation or production ofalternative complement pathway-dependent activation products, includingin conditions involving initiation of the classical complement pathway.

[0126] Seven commercially available anti-human properdin monoclonalantibodies were screened for blocking activity: (1) Quidel anti-humanFactor P#1 (A233); (2) Quidel anti-human Factor P#2 (A235); (3) Dako(Santa Barbara, Calif.) anti human Factor P (MO837); (4) Serum Institute(Copenhagen, Denmark) anti-human Factor P (HYB039 Clone 06); (5) SerumInstitute anti-human Factor P (HYB039 Clone 04); (6) Biogenesis (Poole,UK) anti-human Factor P (Clone 10-18) [same as Quidel #1]; andBiogenesis anti-human Factor P (Clone 10-24) [same as Quidel #2]. Eachof these seven antibodies were able to bind to properdin with highaffinity (K_(D)≈0.1-1 nM). However, only the Quidel P#1 monoclonalantibody (and the identical monoclonal antibody (Clone 10-18) fromBiogenesis) completely blocked alternative pathway complementactivation, as detected by complete inhibition of MAC formation. TheSerum Institute HYB039 clone 04 was found to only partially block andincreasing the concentration of this monoclonal antibody did not achievecomplete blocking. This partially blocking monoclonal antibody and thecompletely blocking Quidel #1 monoclonal antibody have comparablebinding affinities for properdin (K_(D)≈0.1-0.2 nM). According to thepresent invention, agents are therefore effectively screened foressentially complete, partial or no blocking activity in one or moreassays as described herein, including blocking of C3b binding (Example1), blocking of C3bBb binding (Example 2), blocking of alternativepathway-dependent MAC formation (Examples 3 and 5-7), blocking ofalternative pathway-dependent hemolysis (Example 4), blocking ofalternative pathway-dependent C3a formation (Examples 5-7), or blockingof one or more markers of alternative pathway-dependent cell activation(Example 7), including markers of leukocyte activation (e.g.,elastase-antitrypsin, CD11b/CD18), platelet activation (e.g.,P-selection, GPIIIa, GPIb (CD45b), GPIIb) and platelet-leukocyteadhesion. Agents may be further screened for lack of activation of Fcγreceptors and/or classical pathway activation (Example 6).

What is claimed is:
 1. A process of inhibiting alternative complementactivation comprising inhibiting properdin-induced stabilization of C3convertase.
 2. The process of claim 1 wherein the properdin-inducedstabilization of C3 convertase is inhibited by inhibiting the binding ofproperdin to C3b.
 3. The process of claim 2 wherein the binding ofproperdin of C3b is inhibited by exposing properdin to an effectiveamount of an anti-properdin antibody.
 4. The process of claim 3 whereinthe C3b is in the form of C3bBb.
 5. The process of claim 3 wherein theanti-properdin antibody is a monoclonal antibody.
 6. The process ofclaim 3 wherein the C3b is located in the plasma or interstitial fluidof a subject.
 7. The process of claim 6 wherein the subject is a humansubject.
 8. The process of claim 7 wherein the human subject'scomplement has been activated.
 9. The process of claim 8 whereincomplement activation is via an acute pathological injury such asmyocardial infarction, acute respiratory distress syndrome, burn injury,stroke, pancreatitis, cardiopulmonary bypass, or ischemia/reperfusioninjury.
 10. The process of claim 8 wherein complement activationcontributes to a chronic condition such as multiple sclerosis,rheumatoid arthritis, myasthenia gravis or Alzheimer's disease.
 11. Theprocess of claim 6 wherein the anti-properdin antibody is administeredinto the plasma or interstitial fluid of the subject.
 12. A process ofinhibiting the adverse effects of alternative complement pathwayactivation in a subject comprising administering to the subject anamount of an anti-properdin agent effective to selectively inhibitformation of an alternative complement pathway activation product. 13.The process of claim 12 wherein of the alternative complement pathwayactivation product is MAC.
 14. The process of claim 12 wherein thealternative complement pathway activation product is C3a or C5a.
 15. Theprocess of claim 12 wherein the anti-properdin agent is ananti-properdin antibody.
 16. The process of claim 12 wherein theanti-properdin agent is an antigen-binding fragment of an anti-properdinantibody.
 17. The process of claim 12 wherein the anti-properdin agentis a properdin-derived peptide.
 18. The process of claim 12 wherein theanti-properdin agent lacks the ability to activate Fcγ receptors.
 19. Aprocess for inhibiting the adverse effects of classical complementpathway activation in a subject in which the classical complementpathway is initiated comprising administering to the subject an amountof an anti-properdin agent effective to selectively inhibit formation ofan alternative complement pathway activation product.
 20. The process ofclaim 19 wherein the alternative complement pathway activation productis MAC.
 21. The process of claim 19 wherein the alternative complementpathway activation product is C3a or C5a.
 22. The process of claim 19wherein the anti-properdin agent is an anti-properdin antibody.
 23. Theprocess of claim 19 wherein the anti-properdin agent is anantigen-binding fragment of an anti-properdin antibody.
 24. The processof claim 19 wherein the anti-properdin agent is a properdin-derivedpeptide.
 25. The process of claim 19 wherein the anti-properdin agentlacks the ability to activate Fcγ receptors.
 26. A process forinhibiting the adverse effects of classical complement pathwayactivation in a subject in which the classical complement pathway isinitiated comprising administering to the subject an amount of an agentthat inhibits alternative pathway C3 convertase effective to selectivelyinhibit formation of an alternative complement pathway activationproduct.
 27. The process of claim 26 wherein the alternative complementpathway activation product is MAC.
 28. The process of claim 26 whereinthe alternative complement pathway activation product is C3a or C5a. 29.The process of claim 26 wherein the agent is an anti-properdin antibody.30. The process of claim 26 wherein the agent is an antigen-bindingfragment of an anti-properdin antibody.
 31. The process of claim 26wherein the agent is a properdin-derived peptide.
 32. The process ofclaim 26 wherein the agent lacks the ability to activate Fcγ receptors.33. A process for performing a medical procedure on a subjectcomprising: (a) passing circulating blood from a blood vessel of thesubject, through a conduit, and back to a blood vessel of the subject,the conduit having a luminal surface comprising a material capable ofcausing at least one of complement activation, platelet activation,leukocyte activation, or platelet-leukocyte adhesion in the subject'sblood; and (b) introducing an anti-properdin agent into the subject'sbloodstream in an amount effective to reduce at least one of complementactivation, platelet activation, leukocyte activation, orplatelet-leukocyte adhesion resulting from passage of the circulatingblood through the conduit, wherein step (a) occurs before and/or duringand/or after step (b).
 34. The process of claim 33 wherein theanti-properdin agent reduces the alternative pathway-dependentconversion of complement component C3 into complement components C3a andC3b.
 35. The process of claim 33 wherein the anti-properdin agentreduces the alternative pathway-dependent formation of C5b-C9.
 36. Theprocess of claim 33 wherein the anti-properdin agent reduces thealternative pathway-dependent leukocyte activation.
 37. The process ofclaim 33 wherein the anti-properdin agent specifically binds toproperdin and inhibits alternative pathway C3 convertase.
 38. Theprocess of claim 33 wherein the medical procedure is an extracorporealcirculation procedure.
 39. The process of claim 38 wherein theextracorporeal circulation procedure is a cardiopulmonary bypassprocedure.
 40. The process of claim 33 wherein the anti-properdin agentis an anti-properdin antibody.
 41. The process of claim 33 wherein theanti-properdin agent is an antigen-binding fragment of an anti-properdinantibody.
 42. The process of claim 33 wherein the anti-properdin agentis a properdin-derived peptide.
 43. The process of claim 33 wherein theanti-properdin agent lacks the ability to activate Fcγ receptors.
 44. Anarticle of manufacture comprising packaging material and apharmaceutical agent contained within the packaging material, wherein:(a) the pharmaceutical agent comprises an anti-properdin agent, theanti-properdin agent being effective for reducing at least one ofcomplement activation, platelet activation, leukocyte activation, orplatelet adhesion caused by passage of circulating blood from a bloodvessel of a subject, through a conduit, and back to a blood vessel ofthe subject, the conduit having a luminal surface comprising a materialcapable of causing at least one of complement activation, plateletactivation, leukocyte activation, or platelet-leukocyte adhesion in thesubject's blood; and (b) the packaging material comprises a label whichindicates that the pharmaceutical agent is for use in association withan extracorporeal circulation procedure.
 45. The article of manufactureof claim 44 wherein the label indicates that the pharmaceutical agent isfor use in association with a cardiopulmonary bypass procedure.
 46. Useof an anti-properdin agent in the preparation of a medicament forselectively inhibiting formation of an alternative complement pathwayactivation product in a subject.
 47. The use of claim 46 wherein thealternative complement pathway activation product is MAC.
 48. The use ofclaim 46 wherein the alternative complement pathway activation productis C3a or C5a.
 49. The use of claim 46 wherein the anti-properdin agentis an anti-properdin antibody.
 50. The use of claim 46 wherein theanti-properdin agent is an antigen-binding fragment of an anti-properdinantibody.
 51. The use of claim 46 wherein the anti-properdin agent is aproperdin-derived peptide.
 52. The use of claim 46 wherein theanti-properdin agent lacks the ability to activate Fcγ receptors. 53.Use of an anti-properdin agent in the preparation of a medicament forselectively inhibiting formation of an alternative complement pathwayactivation product in a subject in which the classical complementpathway is initiated.
 54. The use of claim 53 wherein the alternativecomplement pathway activation product is MAC.
 55. The use of claim 53wherein the alternative complement pathway activation product is C3a orC5a.
 56. The use of claim 53 wherein the anti-properdin agent is ananti-properdin antibody.
 57. The use of claim 53 wherein theanti-properdin agent is an antigen-binding fragment of an anti-properdinantibody.
 58. The use of claim 53 wherein the anti-properdin agent is aproperdin-derived peptide.
 59. The process of claim 53 wherein theanti-properdin agent lacks the ability to activate Fcγ receptors. 60.Use of an alternative pathway C3 convertase-inhibiting agent in thepreparation of medicament for selectively inhibiting formation of analternative complement pathway activation product in a subject in whichthe classical complement pathway is initiated.
 61. The use of claim 60wherein the alternative complement pathway activation product is MAC.62. The use of claim 60 wherein the alternative complement pathwayactivation product is C3a.
 63. The use of claim 60 wherein the agent isan anti-properdin antibody.
 64. The use of claim 60 wherein the agent isan antigen-binding fragment of an anti-properdin antibody.
 65. The useof claim 60 wherein the agent is a properdin-derived peptide.
 66. Theuse of claim 60 wherein the agent lacks the ability to activate Fcγreceptors.
 67. A pharmaceutical composition comprising (a) ananti-properdin antibody or an antigen-binding fragment of ananti-properdin antibody that (1) selectively inhibits formation of analternative complement pathway activation product; and (2) does notsubstantially activate Fcγ receptors, and (b) a pharmaceuticallyacceptable carrier.
 68. The pharmaceutical composition of claim 67wherein the alternative complement pathway activation product is MAC.69. The pharmaceutical composition of claim 67 wherein the alternativecomplement pathway activation product is C3a or C5a.
 70. Thepharmaceutical composition of claim 67 wherein the anti-properdinantibody is a monoclonal anti-properdin antibody.
 71. The pharmaceuticalcomposition of claim 70 wherein the monoclonal anti-properdin antibodyhas the human γ4 IgG isotype.
 72. The pharmaceutical composition ofclaim 67 wherein the antigen-binding fragment of the anti-properdinantibody is (Fab)₂.
 73. An anti-properdin agent comprising ananti-properdin antibody or an antigen-binding fragment of ananti-properdin antibody that (a) selectively inhibits formation of analternative complement pathway activation product, and (b) does notsubstantially activate Fcγ receptors.
 74. The anti-properdin agent ofclaim 73 wherein the alternative complement pathway activation productis MAC.
 75. The anti-properdin agent of claim 73 wherein the alternativecomplement pathway activation product is C3a or C5a.
 76. Theanti-properdin agent of claim 73 wherein the anti-properdin agent is amonoclonal antibody.
 77. The anti-properdin agent of claim 76 whereinthe monoclonal anti-properdin antibody has the human γ4 IgG isotype. 78.The anti-properdin agent of claim 73 wherein the antigen-bindingfragment of the anti-properdin antibody is (Fab)₂.
 79. A method ofscreening for and selecting an anti-properdin agent comprising (a)assaying the agent for its ability to inhibit formation of analternative complement pathway activation product; (b) assaying theagent for its ability to activate Fcγ receptors; and (c) selecting theagent that inhibits the formation of an alternative complement pathwayactivation product and does not substantially activate Fcγ receptors.80. The method of claim 79 wherein the alternative complement pathwayactivation product is MAC.
 81. The method of claim 79 wherein thealternative complement pathway activation product is C3a or C5a.
 82. Themethod of claim 79 wherein the anti-properdin agent is a monoclonalantibody.
 83. The method of claim 82 wherein the monoclonalanti-properdin antibody has the human γ4 IgG isotype.
 84. The method ofclaim 79 wherein the antigen-binding fragment of the anti-properdinantibody is (Fab)₂.